Methods for treatment of melanoma

ABSTRACT

Embodiments of the present invention are directed to methods for treatment of melanoma using an inhibitor of dihydroorotate dehydrogenase (DHODH) and to combination therapies that involve administering to a subject an inhibitor of oncogenic BRAF (e.g. BRAF(V600E)), as well as an inhibitor of dihydroorotate dehydrogenase (DHODH). Assays for identifying compounds useful for the treatment of melanoma are also provided. The methods comprise screening for compounds or agents that inhibit neural crest progenitor formation in a zebra fish model of melanoma.

CROSS REFERENCE TO RELATED APPLICATIONS

This Application is a Continuation-in Part Application of U.S. Ser. No.13/983,090, filed Oct. 4, 2013, which is a 35 U.S.C. 371 National StageEntry Application of International Application No. PCT/US2012/024295filed Feb. 8, 2012, which designates the U.S. and claims the benefitunder 35 U.S.C. § 119(e) of U.S. Provisional Application No. 61/440,475filed Feb. 8, 2011, the contents of which are incorporated herein byreference in their entireties.

GOVERNMENT SUPPORT

This invention was made with Government support under Grant No.R01CA103846-09 awarded by the National Institute of Health. Thegovernment has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 12, 2016, isnamed 701039-064585-CIP_SL.txt and is 25,804 bytes in size.

FIELD OF INVENTION

The present invention relates to methods for treatment of melanoma usingan inhibitor of dihydroorotate dehydrogenase (DHODH) and to combinationtherapies that involve administering to a subject an inhibitor ofoncogenic BRAF (e.g. BRAF(V600E)), as well as an inhibitor ofdihydroorotate dehydrogenase (DHODH). Assays for identifying compoundsuseful for the treatment of melanoma are also provided. The methodscomprise screening for compounds or agents that inhibit neural crestprogenitor formation in a zebra fish model of melanoma.

BACKGROUND OF THE INVENTION

Melanoma is a malignant tumor of melanocytes. Primarily melanoma is askin tumor, but it is also seen, though less frequently, in themelanocytes of the eye (uveal melanoma). Even though it represents oneof the rarer forms of skin cancer, melanoma underlies the majority ofskin cancer-related deaths and despite many years of intensivelaboratory and clinical research, there are still limited treatments formelanoma.

One effective cure for melanoma (prior to metastasis) is surgicalresection of the primary tumor before it achieves a thickness of greaterthan 1 mm. If the tumor is more invasive, surgery can be combined withradiation and/or chemotherapy. Since these conventional modalitiescannot cure patients of lethal metastasized tumors, efficacy ofalternative treatments such as immunotherapy are being investigated inclinical trials.

Oncogenic BRAF mutations are present in a majority of melanomas and havebeen implicated in malignant growth of melanoma cells. BRAF(V600E)mutation is the most common oncogenic BRAF mutation found in melanomacells. Recently, Zelboraf™ (also known as Vemurafenb or PLX4032:Hoffman-La-Roche (Madison Wis.)/Daiichi Sankyo (Parsippany, N.J.)) wasapproved for treatment of unresectable (inoperable) or metastaticmelanoma with a BRAF(V600E) mutation. There have been positive resultswith Zelboraf™ (Hoffman-La-Roche (Madison Wis.)/Daiichi Sankyo(Parsippany, N.J.)), however resistance is a problem. In addition, thereare unwanted side effects, including back pain, constipation, cough,diarrhea, dizziness, dry skin, hair loss, headaches, joint or musclepain, loss of appetite; nausea, taste changes, thickening of the skin,tiredness, vomiting, and weakness, as well as severe allergic reactions.Thus, treatments for melanoma still need to be improved.

SUMMARY

We have determined by gene expression analysis that melanoma cells adopta fate similar to multipotent neural crest progenitors. We usedzebra-fish embryos to identify the initiating transcriptional eventsthat occur upon activation of oncogenic BRAF(V600E) in the neural crestlineage. Zebrafish embryos that are transgenic for mitfa:BRAF(V660E) andwhich lack p53 have been found to have a gene signature that is enrichedfor markers of multipotent neural crest cells, and neural crestprogenitors from these embryos fail to terminally differentiate. Inparticular, we discovered that BRAF(V600E):p53^(−/−) embryos exhibit anabnormal expansion in the number of crestin⁺ progenitors. Furthermore,in the adult, BRAF(V600E):p53^(−/−) virtually all tumor cells, but nonormal cells, are positive for crestin. Thus, we concluded thatsuppressors of neural crest progenitors may have utility in thetreatment of melanoma. Accordingly, we developed a screen to identifyinhibitors of the crestin⁺ lineage during development, allowing foridentification of compounds or agents useful for melanoma treatment. Wescreened a library of 2,000 chemicals for suppressors of neural crestdevelopment in BRAF(V600E):p53^(−/−) zebrafish and successfullyidentified compounds useful in the treatment of melanoma.

Embodiments of the invention are based on the development of a screeningassay for inhibitors of melanoma, and on the discovery of one class ofcompounds, i.e. inhibitors of dihydroorotate dehydrogenase (DHODH), thatresult in almost complete abrogation of neural crest development inzebrafish. In particular, we have determined that inhibitors of DHODH,when used alone or in combination with an inhibitor of the BRAF oncogene(e.g. BRAF(V600E)), decrease melanoma growth both in vitro and in vivoin mouse xenograft studies. The combination therapy required onlysubclinical doses of each compound suggesting that there may betherapeutic synergy. Unexpectedly, 40% of the treated mice had completeregression of tumors using a combination of the specific oncogenicBRAF(V600E) inhibitor PLX4720 (Plexxikon Inc., Berkeley, Calif., USA)and the DHODH inhibitor Leflunomide (Arava™ Sanofi-Aventis (Paris,France), which led abrogation of tumor growth in nude mice transplantedwith A375 human melanoma cells.

We have also determined that many other inhibitors of DHODH, e.g. whentested in zebra fish, on human A375 melanoma cells and in primary tumorxenographs, when used alone or in combination with an inhibitor of theBRAF oncogene (e.g. BRAF(V600E)), decrease melanoma growth both in vitroand in vivo. In particular, see the compounds of FIG. 21, FIG. 22 andFIG. 23, which are labeled 1-10 and Example III.

Accordingly, provided herein are methods for treatment of melanomacomprising administrating to a subject (diagnosed with, or at risk ofhaving melanoma), an inhibitor of DHODH. Also provided is a combinationtherapy for treatment of melanoma that involves administrating aneffective amount of an inhibitor of DHODH and an effective amount of aninhibitor of oncogenic BRAF (e.g. BRAF(V600E) oncogene), where theinhibitors are administered simultaneously, or sequentially. Methods ofscreening for agents that inhibit melanoma growth are also described.

In one aspect of the invention, a method for treating melanoma in asubject is provided, which comprises administering to a subject in needthereof a therapeutically effective amount of an inhibitor ofdihydroorotate dehydrogenase (DHODH) (e.g. a small molecule, a nucleicacid RNA, a nucleic acid DNA, a protein, a peptide, or an antibody). Inone embodiment, the inhibitor of dihydroorotate dehydrogenase (DHODH) isselected from the group consisting of: leflunomide, teriflunomide,brequinar, dichloroallyl lawsone, maritimus, redoxal and NSC210627, or aderivative thereof. In one embodiment, the DHODH inhibitor is selectedfrom the group consisting of C₁₉H₁₄F₂N₂O₃, compound 1; C₂₀H₁₆F₂N₂O₃,compound 2; C₁₈H₁₃F₆NO₄, compound 3; C₁₉H₁₆F₃NO₄, compound 4;C₁₉H₁₀F₅NO₄S, compound 5; C₁₉H₁₄FNO₄S, compound 6; C₂₀H₁₅F₄NO₄, compound7; C₁₉H₁₇NO₄, compound 8; C₂₀H₁₇F₂NO₄, compound 9; and C₂₀H₁₈FNO₄,compound 10, each as depicted in FIG. 21, FIG. 22 and FIG. 23.

In another aspect of the invention, a combination therapy for treatingmelanoma is provided. The method comprises administering to a subject inneed thereof a therapeutically effective amount of an inhibitor ofdihydroorotate dehydrogenase (DHODH) and an effective amount of aninhibitor of oncogenic BRAF. The inhibitors may be a small molecule, anucleic acid RNA, a nucleic acid DNA, a protein, a peptide, or anantibody. The inhibitors (compounds/agents) may be administeredsimultaneously, or sequentially. In one embodiment, each inhibitor isadministered within minutes, within hours, or within days, of oneanother.

Any oncogenic BRAF can be inhibited, e.g. in one embodiment, theoncogenic BRAF that is inhibited is BRAF(V600E). Alternative oncogenicBRAFs are described within the specification. The inhibitor may bespecific for a particular oncogenic BRAF mutation or alternatively maygenerally inhibit multiple BRAF mutations, and/or wild type BRAF.

Any inhibitor of dihydroorotate dehydrogenase (DHODH) may be used. Inone embodiment, the inhibitor of dihydroorotate dehydrogenase (DHODH) tobe used in combination with an inhibitor of oncogenic BRAF is selectedfrom the group consisting of: leflunomide, teriflunomide, brequinar,dichloroallyl lawsone, maritimus, redoxal and NSC210627, or a derivativethereof. In one embodiment, the DHODH inhibitor is selected from thegroup consisting of C₁₉H₁₄F₂N₂O₃, compound 1; C₂₀H₁₆F₂N₂O₃, compound 2;C₁₈H₁₃F₆NO₄, compound 3; C₁₉H₁₆F₃NO₄, compound 4; C₁₉H₁₀F₅NO₄S, compound5; C₁₉H₁₄FNO₄S, compound 6; C₂₀H₁₅F₄NO₄, compound 7; C₁₉H₁₇NO₄, compound8; C₂₀H₁₇F₂NO₄, compound 9; and C₂₀H₁₈FNO₄, compound 10, each asdepicted in FIG. 21, FIG. 22 and FIG. 23.

In one embodiment, the inhibitor of oncogenic BRAF is selected from thegroup consisting of: Sorafenib, RAF265, XL281, AZ628, GSK2118436,GDC-0879, PLX4032, and PLX4720, or a derivative thereof.

In one embodiment, the inhibitor of oncogenic BRAF is PLX4032 and theinhibitor of dihydroorotate dehydrogenase (DHODH) is leflunomide, or aderivative thereof.

In another embodiment, the inhibitor of oncogenic BRAF is PLX4720 andthe inhibitor of dihydroorotate dehydrogenase (DHODH) is leflunomide, ora derivative thereof.

In another embodiment, the methods of treatment described herein,further comprise selecting a subject that has, or is at risk of havingmelanoma, e.g. a melanoma that expresses oncogenic BRAF. In oneembodiment, the subject has a melanoma that expresses an oncogenic BRAFcomprising a mutation in BRAF selected from the group consisting of:VAL600GLU, ARG461ILE, ILE462SER, GLY463GLU, and LYS600GLU, GLY465VAL andLEU596ARG, and GLY468ARG, GLY468ALA and ASP593GLY.

The methods of the present invention can be used either alone, or inconjunction with other treatment methods known to those of skill in theart. For example, such methods may include, but are not limited to,chemotherapy, radiation therapy, or surgery.

Administration of the inhibitors can be performed by intravenous,intramuscular, subcutaneous, intradermal, topical, intraperitoneal,intrathecal, intrapleural, intrauterine, rectal, vaginal, intrasynovial,intraocular/periocular, intratumor or parenteral administration.

In one embodiment, the subject is at risk for developing melanoma andthe combination therapy, or DHODH inhibitor, is administeredprophylactically. The risk can be determined genetically. Alternatively,the risk can be determined by measuring levels of marker proteins in thebiological fluids (i.e. blood, urine) of a patient. In one embodiment,the methods of treatment described herein further comprises the step ofselecting a subject in need thereof of treatment, e.g. selecting asubject diagnosed with melanoma, or a subject at increased risk ofmelanoma (e.g. potential cancer relapse).

In another aspect of the invention, screening methods for identifyingagents that inhibit melanoma growth are provided. The methods comprise,(a) contacting a zebrafish embryo with a test agent for a period oftime, (b) rinsing the test agent from the embryos of step (a); and (c)assaying the number of neural crest progenitors as compared to a controlzebrafish embryo that has not been contacted with the test agent,wherein a reduced number of neural crest progenitors (e.g. and theirdifferentiation into melanocytes) indicates that the compound is capableof inhibiting melanoma. The zebrafish embryos may be wild type zebrafish embryos or transgenic zebrafish embryos.

In one embodiment, the number of neural crest progenitors is assayed bymonitoring crestin expression, or sox10 expression, or dct expression,e.g. by quantitation of ISH studies.

In one embodiment, the transgenic zebrafish embryo expresses greenfluorescent protein operably linked to the melanocyte mitfa promoter andmelanocyte neural crest progenitors are monitored by GFP expression.

BRIEF DESCRIPTION OF FIGURES

FIGS. 1a to 1c show zebrafish melanoma and neural crest gene expression.FIG. 1a , Transgenic zebrafish expressing BRAF^(V600E) under themelanocyte specific mitfa promoter develop pigmentation abnormalities,and melanoma when crossed with p53^(−/−) fish. Gross embryonicdevelopment is largely normal. FIG. 1b , Gene expression analysisreveals a unique gene signature at 72 hpf in the BRAF^(V600E);p53^(−/−)strain (left). Gene set enrichment analysis (GSEA) reveals an enrichmentbetween the embryonic gene signature and the adult melanomas which form4-12 months later (middle and right; see Methods for full GSEA methods).Embryo heat map columns represent average of 3 clutches (log₂ scale,range −2 to +2 fold); adult heat map columns represent individual fish(log₂ scale, range −10 to +10 fold). FIG. 1c , Sagittal section of WTand BRAF^(V600E);p53^(−/−) adults reveal homogeneous crestin expression(blue staining, shown as dark grey) only within the dorsal melanoma,whereas it is absent in normal adult tissues.

FIGS. 2a to 2b show ISH staining in Zebrafish. A chemical genetic screento identify suppressors of neural crest development. FIG. 2a , Achemical genetic screen to identify suppressors of the crestin⁺ lineageduring embryogenesis identified NSC210627, a compound which completelyabrogates expression by ISH (FIG. 2a , top and middle). The Discoverygate chemoinformatic algorithm revealed structural similarity betweenNSC210627 and brequinar (see FIG. 5), an inhibitor of dihydroorotatedehydrogenase (DHODH). Leflunomide, FIG. 2a structurally distinct DHODHinhibitor, phenocopies the crestin phenotype of NSC210627 (FIG. 2a ,right). FIG. 2a bottom panel; Leflunomide caused an absence of multipleneural crest derivatives, including pigmented melanocytes, mitf-GFP⁺melanocyte progenitors, and mbp-mCherry⁺ glial cells. FIG. 2b ,Leflunomide or A771726 (see FIG. 6a ) significantly reduced the numberof multipotent daughter cells that could be subcloned from individualprimary neural crest stem cell colonies (Values shown are mean+/−SD ofn=3 replicates; *, p<0.05 compared to control, t-test).

FIGS. 3a to 3c show that DHODH inhibition modulates transcriptionalelongation. FIG. 3a , The hypomorphic spt5^(m806) mutant has only a mildpigment defect on its own (top). Treatment with low-dose leflunomide (3uM) leads to an almost complete absence of neural crest derivedmelanocytes in the mutant line. See FIG. 7 for dose-responsequantification of this effect. FIG. 3b , Metagene analysis of RNA pol IIoccupancy in A375 human melanoma cells after treatment with leflunomide.Pol II occupancy at the promoter region is unaffected, but diminished atthe 3′ end of the genes. Inset shows a higher magnification of the 3′region of the genes. FIG. 3c , Representative examples of myc targetgenes which demonstrate defects in transcriptional elongation afterleflunomide treatment, along with a non-affected gene. For Npm1, the TRin DMSO=5.04, and in LEF=8.10. For Ccnd1, the TR in DMSO=3.47, and inLEF=4.67.

FIGS. 4a to 4c show graphs. DHODH blockade suppresses melanoma growth inconcert with BRAF^(V600E) inhibition. FIG. 4a , Leflunomide causes adose-dependent decrease in melanoma proliferation as measured byCellTiterGlo assay in 3 BRAF^(V600E) melanoma cells lines tested (A375,RPMI7951, Hs.294T). FIG. 4b , FIG. 4c Leflunomide cooperates with theBRAF^(V600E) inhibitor PLX4720 in inhibiting melanoma cell proliferationin the A375 (FIG. 4b ) and Hs.294T (FIG. 4) cell lines as well as theother tested lines (See FIG. 8). FIG. 4d , After subcutaneoustransplantation of A375 cells (3×10⁵) into nude mice, both leflunomideand PLX4720 impair tumor progression, with the combination showing anearly complete abrogation of tumor growth and in 2/5 animals completetumor regression. (*p=0.036 DMSO vs. PLX; **p=0.006 DMSO vs. LEF;***p=0.006 PLX or LEF vs. PLX/LEF; PLX vs. LEF: p=NS, ANOVA followed byTukey post-hoc analysis). Values shown are mean+/−SEM of n=3-5replicates, as shown.

FIG. 5 shows chemical structures of DHODH inhibitors NSC210627,brequinar, leflunomide and A771726 (teriflunomide) and oncogenic BRAFinhibitor PXL4720.

FIGS. 6a to 6c : The effects the leflunomide derivative A771726, on ratneural crest stem cells. FIG. 6a , The effects of A771726 on rat neuralcrest stem cell self-renewal (as described in FIG. 2, *, p<0.05,compared to control, t-test). FIG. 6b , FIG. 6c , Both compounds causeno defect in plating efficiency or affect individual progeny (NGM=nerve,glia, smooth muscle) without (N, G, or M colonies) survival ordifferentiation capacity (p>0.05, n=3, ANOVA). Values shown aremean+/−SD of n=3 replicates.

FIGS. 7a to 7d show graphs of the effect of low-doses of leflunomide onpigmentation in the spt5_(m806) hypomorphic mutant. Quantification ofthe effect described in FIG. 3a , in which 3, 4 or 5 uM leflunomide wasapplied to a spt5^(m806) hypomorphic incross. FIG. 7a , Pigmentationscores in untreated embryonic offspring of an incross of the spt5^(m806)hypomorphic mutant. There is a mild pigmentation defect only inhomozygous embryos. FIG. 7b , FIG. 7c , FIG. 7d Leflunomide at 3, 4 or 5uM demonstrates that both heterozygous and homozygous mutants embryosshow increased sensitivity to pigment loss when compared to wild-type(*, p=0.000018, Kruskal-Wallis, n values as indicated in Figure).

FIGS. 8a to 8d show graphs. FIG. 8a , FIG. 8 ab, In vitro proliferationassay testing the effects of combined A771726 and the BRAF inhibitorPLX4720 on RPMI7951 and 294T cells shows significant augmentation of theeffects of the BRAF inhibitor alone. FIG. 8c , PLX4720 is only effectivein BRAF^(V600E) melanoma cells. Although RPMI7951 is BRAF^(V600E), it issignificantly less sensitive than the other tested cell lines. FIG. 8d ,Proliferation in the presence of A771726 in pancreatic cancer (8988,PANC1) and breast cancer (MDA-MD-231) cell lines. Values shown aremean+/−SEM of n=4 replicates.

FIGS. 9a to 9b , show a graph of a human melanoma tissue microarray(n=70 samples) which was analyzed for markers of neural crestprogenitors (ednrb, edn3) and melanocyte progenitors (mitf, dct). Themajority of human melanomas are positive for neural crest markers butonly a fraction express more differentiated markers. FIG. 9b , showsmarker staining of tumors, a representative tumor in which all 4 markersare expressed in different areas of the tumor.

FIG. 10 shows cumulative results of the chemical screen described in theMethods section. 2000 chemicals were screened, and positive “hits” werethose with a staining score of less than 2 (i.e. 3%). Representativeexamples of each score are shown on top. The percentage of chemicalsresulting in a given score are shown below.

FIGS. 11 shows graphs depicting enzymatic inhibition. The effect ofNSC210627 on purified human and malarial DHODH. This demonstrates astrong inhibition of both enzymes, at lower concentrations in the humanversus malarial forms. Error bars are mean+/−SEM of n=3 replicates.

FIG. 12 shows in situ hybridization for neural crest and non-neuralcrest lineages in the presence of leflunomide. No significant differenceis seen in intensity for the mesoderm marker nt1 (although the patternof expression is altered in some embryos) and blood marker gata1. Adisruption in the pattern of foxd3 expression, and absence of sox10 anddct expression confirms broad defects in neural crest development in thepresence of leflunomide.

FIGS. 13a to 13c show in situ hybridization and graphs. FIG. 13a , Thespt5sk8−/− transcriptional elongation mutant phenocopies the absence ofcrestin+ progenitors in a manner analogous to leflunomide. FIG. 13b ,GSEA was used to compare the gene expression signature of leflunomidetreated embryos and spt5sk8−/− embryos at 24 hpf. Venn diagram shows thenumber of genes up or downregulated in the spt5sk8−/− mutant that aresimilarly up or downregulated after leflunomide treatment. (see Methodsfor full GSEA analysis methods) FIG. 13c , qRT-PCR on whole embryostreated with DMSO or leflunomide was used to measure transcriptionelongation at the 5′ or 3′ end of the noted gene. Leflunomide treatmentcaused a significant decrease in 3′ transcription of mitf, pvalb2,her4.2 and dlb, whereas control genes showed no bias towards 5′ or 3′effects (n=6 replicates in each group, *p<0.05, two-sided t-test,leflunomide vs. DMSO). Values shown are mean+/−SEM.\

FIGS. 14a to 14b show metagene analysis graphs. ChIP-seq results inmelanoma MAMLE-3M cells. Data are the same as that described in FIG.3b,3c (A375 melanoma cells). FIG. 14a , Metagene analysis of RNApolymerase II occupancy of the promoter and gene body in MAMLE-3M cellstreated with DMSO (control) or leflunomide 50 uM. Leflunomide causes nodefect in promoter occupancy but significantly decreases pol IIoccupancy in the body of the gene, consistent with an inhibition oftranscriptional elongation. FIG. 14b , Representative examples ofaffected (left) and unaffected genes in this cell line.

FIGS. 15a to 15c show a Western blot and graphs. FIG. 15a , Western blotshowing extent of DHODH knockdown induced by shRNA#877 as compared toscrambled (scr) control shRNA in A375 melanoma cells. FIG. 15b , Cellcounts measured over an 8 day period in A375-scr and A375-shRNA#877cells shows a 57% decrease in cellular proliferation with DHODHknockdown. FIG. 15c , ChIP-PCR in A375-scr and A375-shRNA#877 cellsdemonstrates decreased RNA pol II binding at the 3′ end of myc, npml andotub, consistent with an inhibition of transcriptional elongation(*p<0.05, +p=0.1 t-test, A375-sh877 vs. A375-scr cells, n=3-4). Valuesshown are mean+/−SEM.

FIG. 16 show mice after treatment with inhibitors. Representativeexamples of mice after treatment with DMSO, PLX alone, leflunomide aloneor the combination. See FIG. 4 for quantification.

FIG. 17 shows the chemical structures of Sorafenib (Bayer) and RAF265.

FIG. 18 shows the chemical structures of AZ628, and GSK2118463.

FIG. 19 shows the chemical structures of PLX4032 and GDC-0879.

FIG. 20 shows the chemical structures of DHODH inhibitors dichloroallyllawsone, maritumus (see U.S. Pat. No. 7,256,008), and redoxal.

FIG. 21 shows the chemical structures of DHODH inhibitors Compound 1,Compound 2, Compound 3, and Compound 4, which are used in Example III.Synthesis of compound 1 and compound 2 is described in U.S. PatentPublication 2010/007898. Synthesis of compounds 3 and 4 is described inU.S. Pat. No. 7,074,831.

FIG. 22 shows the chemical structures of DHODH inhibitors: Compound 5and Compound 6, which are used in Example III. Synthesis of compound 5and compound 6 is described in U.S. Patent Publication 2004/0192758.

FIG. 23 shows the chemical structures of DHODH inhibitors: Compound 7,Compound 8, Compound 9, and Compound 10, which are used in Example III.Synthesis of these compounds is described in U.S. Patent Publication2006/0199859 and in U.S. Pat. No. 7,176,241.

FIGS. 24a to 24b show chemical structures and in situ hybridization(ISH) staining in Zebrafish; FIG. 24a shows the chemical structures forleflunomide, which gets converted into its active form A771726 alsoshown, which causes dihydroorotate dehydrogenase inhibition. Also shownin FIG. 24a is a structurally different DHODH inhibitor, inhibitor DH#1,also known as Compound 1 herein, as used in Example III. FIG. 24b areISH staining showing the effect of DHODH inhibitor compound 1 (DH#1);leflunomide; and A771726 on neural crest development, in the presence ofDMSO, Decylubiquinone, Aphiadicolin, or Progesterone. FIG. 24b showsthat all of these inhibitors (leflunomide, A77 1726, or DH #1) lead toreduction in expression of the neural crest, depicted by an in situhybridization for the neural crest marker crestin. However, chemicalsincluding decylubiquinone, aphidicolin, and progesterone suppress thesedrugs and lead to a rescue of the neural crest population.

FIGS. 25a to 25b show in situ hybridization (ISH) staining in Zebrafishembryos to monitor neural crest development in the presence of compound1, compound 7, leflunomide, compound 5 and compound 6 (See FIGS. 21-23for compound structure and formula). Different concentrations of theDHODH inhibitors and in situ for crestin revealed compound 1 andcompound 7 (FIG. 25a (#1 and #7) impair crestin expression at lowerdoses compared to leflunomide and compounds 5 and 6, FIG. 25 b #5 and#6, respectively. The compounds showed varied strength in their abilityto inhibit neural crest development, the effect of compound 1>compound7>leflunomide, which is approximately the same as compounds 5 and 6.

FIG. 26 shows in situ hybridization (ISH) staining in Zebrafish embryosto monitor neural crest development in the presence of compound 4,compound 9 and compound 2 (#4, #9 and #2 respectively; See FIGS. 21 to23 for compound structure and formula). When referring also to FIG. 25,it is shown that DHODH inhibitors #4, #9 and #2 are less effective thanleflunomide. Indeed at 1.75 uM these drugs do not affect crestinexpression like observed upon the same dose of leflunomide in FIG. 25 b.

FIG. 27 shows a graph depicting the effect of all of the DHODHinhibitors compounds 1-10 (DHODH analogs) (See FIGS. 21-23 forcompounds) on A375 cells after 3 days of treatment. y-axis percentinhibition, x-axis concentration. Compounds 1, 4, and 7 significantlydecrease cell proliferation at lower doses compared to leflunomide andA771726.

FIGS. 28a to 28e show graphs depicting the effects of leflunomide,Compound 1 (PH-1), and Compound 7 (PH-7) (See FIG. 21 (compound 1) andFIG. 23 (compound 7)) on the indicated melanoma cells in vitro. FIG. 28a, M481 cells. FIG. 28b , A375 cells. FIG. 28c , M405 cells. FIG. 28d ,M528 cells. FIG. 28e , M715 cells. Compound 1, Compound 7 andleflunomide affect cell viability of human primary melanoma cells. FIGS.28a and 28b show respectively that leflunomide as well as DHODH #1(PH-01, i.e. compound 1), DHODH#7 (PH-07, i.e. compound 7) impair cellsurvival in primary human melanoma cells (M481) and in the melanoma cellline (A375). FIG. 28c shows that the effect of these inhibitors isvariable in different primary melanoma cells (M405, M528 and M715).

FIGS. 29a to 29d are graphs showing the in vivo effects of leflunomidein combination with digitoxin on M405 tumor xenografts in mouse. FIG.29a , effect on tumor diameter (cm) vs. time. FIG. 29b , effect on tumorweight. FIG. 29c , effect on frequency of melanoma cells in the blood.FIG. 29d , Total flux (photons/second). M405 were not sensitive to drugtreatment as shown in FIG. 28 and accordingly there is no effect ontumor growth (FIG. 29a ) and number of melanoma cells in bloodcirculating cells (FIG. 29b ). But there is significant difference inblood circulating cells in co-treatment with Leflunomide and Digitoxin.No change in flux was observed (FIG. 29d ).

FIG. 30 is a graph of in vivo effects of the combination of DHODHinhibitor and Braf inhibitor on tumor volume, of M481 primary humanmelanoma xenografts in mice. FIG. 30 shows the effect of leflunomide incombination with Plexicon in xenografts mouse model. M841 primary humanmelanoma xenografts in mice revealed that leflunomide is more effectivewhen used in combination with Plexicon.

FIG. 31 is a graph that shows the effect leflunomide in combination withBraf and Mek inhibitors on primary human melanoma cells xenografts inmice. Human BRAFV600 (M491) cells were treated with a combination ofdrugs including BRAF inhibitors (BRAFi), MEK inhibitors (MEKi) andLeflunomide. Leflunomide significantly impair tumor growth incombination with BRAFi and MEKi.

DETAILED DESCRIPTION

The present invention relates generally to a method of treating melanomain a subject having, or at risk of having, melanoma.

As used herein, the term “subject” or “patient” or refers to any mammal.The patient is preferably a human, but can also be a mammal in need ofveterinary treatment.

The term “melanoma” as used herein includes all types of melanoma,including, for example, melanoma skin cancer, ocular melanoma, andmucosal melanoma. Melanoma is caused by changes melanocytes that producemelanin. There are four major types of melanoma: 1) superficialspreading melanoma, which is usually flat and irregular in shape andcolor, with different shades of black and brown and is most common inCaucasians, 2) nodular melanoma, which usually starts as a raised areathat is dark blackish-blue or bluish-red, but can be colorless, 3)Lentigo maligna melanoma, which usually occurs in the elderly and ismost common in sun-damaged skin on the face, neck, and arms. Theabnormal skin areas are usually large, flat, and tan with areas ofbrown, 4) Acral lentiginous melanoma, which is the least common form andusually occurs on the palms, soles, or under the nails and is morecommon in African Americans. Melanomas may also appear in the mouth,iris of the eye, or retina at the back of the eye and can be foundduring dental or eye examinations. Although very rare, melanoma can alsodevelop in the vagina, esophagus, anus, urinary tract, and smallintestine.

The presence of melanoma can be determined by means well known to thoseof skill in the art, e.g. tissue biopsies and in situ assays in whichmalignant melanoma (malignant melanocytes scattered in all epidermallayers) show atrophic epidermis, prominent dermal solar elastosis andalmost always lymphocytic infiltration. Invasion of the dermis bymelanocytes may occur in lentigo maligna melanoma. In addition, melanomamay be detected by methods that include, but are not limited,immunohistochemistry using the melanoma specific antibody HMB-45, orRT-PCR with different melanoma associated antigens (MAA) including, butnot limited to tyrosinase, MART-1/Melan A, Pmel-17, TRP-1, and TRP-2(see, e.g., Hatta N., et al., J Clin Pathol. 1998 August;51(8):597-601). Biomarkers for melanoma are also known and can be usedfor example to assess subjects at risk of melanoma. Non-limiting examplebiomarkers for melanoma are described in PCT Publications WO2008/141275, WO 2009/073513, or in U.S. Pat. No. 7,442,507, which areherein incorporated by reference in their entirety.

Symptoms of melanoma include, but are not limited to, a mole, sore,lump, or growth on the skin that may bleed, or exhibit change in skincoloring. Often patients are told of an ABCDE system the can help themremember possible symptoms of melanoma to watch out for: Asymmetry: amole where one half of the abnormal area is different from the otherhalf; Borders, the edges of the growth are irregular; Color, the colorchanges from one area to another, with shades of tan, brown, or black,and sometimes white, red, or blue, e.g. a mixture of colors may appearwithin one sore; Diameter, the spot is usually (but not always) largerthan 6 mm in diameter, about the size of a pencil eraser; and Evolution,the mole keeps changing appearance.

As used herein, “treatment”, “treating”, “prevention” or “amelioration”of melanoma refers to inhibition of growth of a melanoma, inhibitingmetastasis of melanoma, delaying or preventing the onset of melanoma, orreversing, alleviating, ameliorating, inhibiting, slowing down, orstopping the progression of melanoma. The term “treatment” as usedherein is not intended encompass 100% cure of melanoma. However, in oneembodiment, the therapeutic methods described herein may result in 100%reversal of disease.

In one embodiment of the methods described herein, at least one symptomof melanoma is alleviated by at least 5%, at least 10%, at least 20%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, or at least 90%. In such an embodiment, the clinical signsand/or the symptoms associated with the melanoma are lessened as aresult of the administration of the inhibitor/s. The signs or symptomsto be monitored are characteristic of a particular melanoma and areknown to the skilled clinician, as well as the methods for monitoringthe signs and conditions.

In one embodiment of the methods described herein, the melanoma lesionsize is reduced by at least 5%, at least 10%, at least 20%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least80%, or at least 90%.

In one embodiment of the methods describe herein, melanoma cellproliferation, or melanoma growth, is inhibited by at least 5%, at least10%, at least 20%, at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 80%, or at least 90%. The skilled clinicianmay monitor the size or rate of growth of a tumor using a diagnosticimaging method typically used for the particular tumor (e.g., usingultrasound or magnetic resonance image (MRI) to monitor a tumor).

In one embodiment, the method for treatment of melanoma comprisesadministering to a subject in need thereof a therapeutically effectiveamount of an inhibitor of DHODH.

As used herein the term “dihydroorotate dehydrogenase (DHODH)” refers tothe enzyme that catalyzes the fourth step in the de novo biosynthesis ofpyrimidine. DHODH converts dihydroorotate to orotate. The sequences ofDHODH are well known to those of skill in the art, e.g. a human DHODHprotein sequence is found at GenBank accession no. AAA50163.1 (SEQ IDNO:2), gene (SEQ ID NO:4). Enzymatic activity of DHODH can be assessedusing in vitro assays and monitoring the reduction in2,6-dichloroindophenol (DCIP) (e.g. see Examples).

Any inhibitor of DHODH can be used in methods of the invention. As usedherein, the phrase “inhibitor of DHODH” means a compound or agent thatinhibits the biological activity of DHODH. The biological activity ofDHODH can be inhibited using a compound or agent that inhibits theenzymatic activity of DHODH, or a compound or agent that down regulatesexpression or availability of DHODH in a cell or organism (e.g. siRNA,shRNA). Many inhibitors of DHODH are known to those skilled in the art.For example, various inhibitors are described in: Leban et al. (2005)SAR, species specificity, and cellular activity of cyclopentenedicarboxylic acid amides as DHODH inhibitors, Bioorganic & MedicinalChemistry Letters 15(21): 4854-4857; and Fritzson et al. (2010)Inhibition of human DHODH by 4-Hydroxycoumarins, fenamic acids, andN-(alkylcarbonyl)anthranilic acids identified by structure-guidedfragment selection Chem Med Chem 5(4): 608-617; Kulkarni et al. (2010)4SC-101 a novel small molecule dihydroorotate dehydrogenase inhibitor,suppresses systemic lupus erythematosus in MRL-(Fas)lpr mice Am JPathol. 176(6):2840-7;

Various DHODH inhibitors have been disclosed for the treatment orprevention of autoimmune diseases, immune and inflammatory diseases,destructive bone disorders, malignant neoplastic diseases,angiogenic-related disorders, viral diseases, and infectious diseases.See for example WO2010083975; WO2011138665; WO200137081; WO2009133379;WO 2009021696; WO200 082691; WO2009029473; WO2009153043; US2009209557;US2009 062318; US2009082374; WO2008097180; W02QQ8Q77639; US2008027079;US2007 299U4; US2007027193; US2007224672; WO2007149211; JP2007015952;WO2006 044741; WO2006001961; WO2006051937; WO2006038606; WO2006022442;US2006 199856; WO2005075410; U.S. Pat. No. 7,074,831; WO2004056797; U.S.Pat. No. 7,247,736; WO2004056747; WO 2004056746; JP2004099586;WO2003097574; WO2003030905; WO2003006425; WO2003 006424; US2003203951;WO2002080897; U.S. Pat. No. 7,176,241; U.S. Pat. No. 7,423,057;WO2001024785; U.S. Pat. No. 6,841,561; WO9945926; WO9938846; WO9941239;EP767167 and U.S. Pat. No. 5,976,848, each of which are hereinincorporated by reference in their entirety. For additional reviews andliterature regarding DHODH inhibitors see Bio & Med. Chem. Letters,20(6), 2010, pages 1981-1984; Med. Chem. 2009, 52, 2683-2693; J. Med.Chem. 2008, 51 (12), 3649-3653.

For example, inhibitors include, but are not limited to, leflunomide,teriflunomide, brequinar (NSC 368390) (Cancer Research 1992, 52,3521-3527), Dichloroallyl lawsone (The Journal of Biological Chemistry1986, 261(32), 14891-14895; McKelvey et al. Clin Pharmacol Ther. 1979May; 25(5 Pt 1):586-90.), Maritimus (F 778) (Drugs of the Future 2002,27(8), 733-739) and Redoxal (The Journal of Biological Chemistry 2002,277(44), 41827-41834). See also Example 1 and FIG. 5, which describes acompound of previously unknown function, NSC210627, with similarity tobrequinar, a known inhibitor of DHODH.

In one embodiment, the DHODH inhibitor is selected from the groupconsisting of C₁₉H₁₄F₂N₂O₃, compound 1; C₂₀H₁₆F₂N₂O₃, compound 2;C₁₈H₁₃F₆NO₄, compound 3; C₁₉H₁₆F₃NO₄, compound 4; C₁₉H₁₀F₅NO₄S, compound5; C₁₉H₁₄FNO₄S, compound 6; C₂₀H₁₅F₄NO₄, compound 7; C₁₉H₁₇NO₄, compound8; C₂₀H₁₇F₂NO₄, compound 9; and C₂₀H₁₈FNO₄, compound 10, each asdepicted in FIG. 21, FIG. 22 and FIG. 23. Compound 1, Compound 2,Compound 3, and Compound 4, which are used in Example III. Synthesis ofcompound 1 and compound 2 is described in U.S. Patent Publication2010/007898, which is herein incorporated by reference in its entirety.Synthesis of compounds 3 and 4 is described in U.S. Pat. No. 7,074,831,which is herein incorporated by reference in its entirety. FIG. 22 showsthe chemical structures of DHODH inhibitors: Compound 5 and Compound 6,which are used in Example III. Synthesis of compound 5 and compound 6 isdescribed in U.S. Patent Publication 2004/0192758, which is hereinincorporated by reference in its entirety. FIG. 23 shows the chemicalstructures of DHODH inhibitors: Compound 7, Compound 8, Compound 9, andCompound 10, which are used in Example III. Synthesis of these compoundsis described in U.S. Patent Publication 2006/0199859 and in U.S. Pat.No. 7,176,241, which are herein incorporated by reference in theirentirety.

Leflunomide is sold under the trade name Arava (EP 0 780 128, WO97/34600), and was the first DHODH inhibitor that reached the marketplace. Leflunomide is the prodrug of teriflunomide (A771726), which isthe active metabolite that inhibits human DHODH with a moderate potency.In one specific embodiment, the inhibitor of DHODH is not leflonomide orits derivative A771726. In one embodiment, the inhibitor of DHODH is notmaritumis.

In one embodiment, the inhibitor of DHODH is not C₁₉H₁₄F₂N₂O₃, compound1 (FIG. 21). In one embodiment, the inhibitor of DHODH is notC₂₀H₁₆F₂N₂O₃, compound 2 (FIG. 21). In one embodiment, the inhibitor ofDHODH is not C₁₈H₁₃F₆NO₄, compound 3 (FIG. 21). In one embodiment, theinhibitor of DHODH is not C₁₉H₁₆F₃NO₄, compound 4 (FIG. 21). In oneembodiment, the inhibitor of DHODH is not C₁₉H₁₀F₅NO₄S, compound 5 (FIG.22). C₁₉H₁₄FNO₄S, compound 6 (FIG. 22). In one embodiment, the inhibitorof DHODH is not C₂₀H₁₅F₄NO₄, compound 7 (FIG. 23). In one embodiment,the inhibitor of DHODH is not C₁₉H₁₇NO₄, compound 8 (FIG. 23). In oneembodiment, the inhibitor of DHODH is not C₂₀H₁₇F₂NO₄, compound 9 (FIG.23). In one embodiment, the inhibitor of DHODH is not C₂₀H₁₈FNO₄,compound 10 (FIG. 23).

Additional compounds or agents that inhibit DHODH expression or activitymay be readily identified using known screening methods. In oneembodiment, the compounds identified binds specifically to the DHODHpolypeptide and inhibit its enzymatic activity. The ability of acompound to inhibit DHODH can be determined using enzymatic assays wellknown to those of skill in the art. Target validation models are knownto those of skill in the art, e.g. Zameitat et al. (2007) FunctionalExpression of Human Dihydroorotate Dehydrogenase (DHODH) in pyr4 Mutantsof Ustilago maydis Allows Target Validation of DHODH Inhibitors In VivoAppl. Environ. Microbiol. 73(10) 3371-3379.

The term “agent” or “compound” as used herein and throughout thespecification means any organic or inorganic molecule, includingmodified and unmodified nucleic acids such as antisense nucleic acids,RNAi, such as siRNA or shRNA, peptides, small molecules peptidomimetics,receptors, ligands, and antibodies. e.g. molecules and/or compositionsthat inhibit DHODH activity or inhibit BRAF activity. Thecompounds/agents include, but are not limited to, chemical compounds andmixtures of chemical compounds, e.g., small organic or inorganicmolecules; saccharines; oligosaccharides; polysaccharides; biologicalmacromolecules, e.g., peptides, proteins, and peptide analogs andderivatives; peptidomimetics; nucleic acids; nucleic acid analogs andderivatives; extracts made from biological materials such as bacteria,plants, fungi, or animal cells or tissues; naturally occurring orsynthetic compositions; peptides; aptamers; and antibodies, or fragmentsthereof.

A compound/agent can be a nucleic acid RNA or DNA, and can be eithersingle or double stranded. Example nucleic acid compounds include, butare not limited to, a nucleic acid encoding a protein inhibitor (e.g.transcriptional inhibitors), oligonucleotides, nucleic acid analogues(e.g. peptide-nucleic acid (PNA), pseudo-complementary PNA (pc-PNA),locked nucleic acid (LNA) etc.), antisense molecules, ribozymes, smallinhibitory or activating nucleic acid sequences (e.g. RNAi, shRNAi,siRNA, micro RNAi (mRNAi), antisense oligonucleotides etc.) A proteinand/or peptide agent can be any protein that modulates gene expressionor protein activity. Non-limiting examples include mutated proteins;therapeutic proteins and truncated proteins, e.g. wherein the protein isnormally absent or expressed at lower levels in the target cell.Proteins can also be selected from genetically engineered proteins,peptides, synthetic peptides, recombinant proteins, chimeric proteins,antibodies, midibodies, minibodies, triabodies, diabodies, humanizedproteins, humanized antibodies, chimeric antibodies, modified proteinsand fragments thereof. A compound or agent that increases expression ofa gene or increases the activity of a protein encoded by a gene is knownas an activator or activating compound. A compound or agent thatdecreases expression of a gene or decreases the activity of a proteinencoded by a gene is also known as an inhibitor or inhibiting compound.

The terms “polypeptide,” “peptide” and “protein” refer to a polymer ofamino acid residues. The terms apply to amino acid polymers in which oneor more amino acid residue is an artificial chemical mimetic of acorresponding naturally occurring amino acid, as well as to naturallyoccurring amino acid polymers and non-naturally occurring amino acids.

In some embodiments, the methods of the invention are directed to acombination therapy wherein one component is administration of aneffective amount of an inhibitor of BRAF oncogene (e.g. BRAF(V006E)),and a second component is administration of an effective amount of aninhibitor of DHODH.

As used herein the term “BRAF” refers to the serine/threonine kinaseBRAF polypeptide as well as the gene encoding it. BRAF is known totransduce regulatory signals from RAS through MEK (MAPK kinase) to ERK.Sequences for BRAF genes and proteins are well known to those of skillin the art, e.g. the protein sequence for human wild type BRAF is foundat GenBank accession no. NP_004324.2 (SEQ ID NO: 1). BRAF oncogenicmutations lead to constitutive activation of the RAS-RAF-mitogenactivated protein kinase/ERK kinase (MEK)-extracellular signal regulatedkinase (ERK) signaling pathway, which is essential for cellproliferation, differentiation and survival (Davies et al., Nature417:949-54 (2002)); English et al., Trends Pharmacol. Sci. 23:40-5(2002)). A complete cDNA for human BRAF is disclosed in Sithanandam, G.,et al. complete coding sequence of a human B-raf cDNA and detection ofB-raf protein kinase with isozyme specific antibodies Oncogene 5 (12),1775-1780 (1990), see also GenBank accession No: NM_004333.4, (SEQ IDNO: 5).

As used herein, “oncogenic BRAF” refers to a BRAF gene/polypeptide thatis aberrantly expressed or aberrantly active. This increase inexpression or activity can result from a mutation(s) in BRAF, whichconstitutively activates the MEK/ERK or other pathways, or from a defectresulting in a detectable increase in BRAF expression or activity incancer cell compared to a non-cancerous. In addition to mutations, suchincreased expression and/or activity may result from amplification of awild-type BRAF nucleic acid, overexpression of a wild-type BRAF protein,e.g., by aberrant regulation of the BRAF regulatory region such as thepromoter, overexpression or activation of BRAF due to aberrantregulation of an upstream regulator (e.g., RAS mutation, or inhibitionof a BRAF inhibitor) or by stabilization of BRAF.

Oncogenic BRAF mutations have been identified in about 70% of malignantmelanomas (Davies et al., Nature 417:949-54 (2002)) and are implicatedin the malignant growth of melanoma cells (Wellbrock et al., Cancer Res.64:2338-42 (2004); Hingorani et al., Cancer Res. 63:5198-202 (2003)).The term “mutation” includes substitution, deletions, inversions,insertions, premature terminations and the like. A T1796A transversionin exon 15, which results in a V600E (also known as V599E) substitutionin the BRAF kinase domain, accounts for about 90% of BRAF mutationsdetected in melanoma samples (Davies et al., supra). The V600E mutationincreases BRAF kinase activity (Davies et al., supra; Dong et al.,Cancer Res. 2003; 63: 3883-3885). Additional mutations of the BRAF genein human cancer, include but are not limited to, ARG461ILE, ILE462SER,GLY463GLU, and LYS600GLU (Rajagopalan, H., et al. (Letter) Nature 418:934, 2002), GLY465VAL and LEU596ARG (Naoki, K., et al., Cancer Res. 62:7001-7003, 2002), and GLY468ARG, GLY468ALA and ASP593GLY (Lee, J. W., etal., Brit. J Cancer 89: 1958-1960, 2003), the references of which areeach incorporated by reference in their entirety.

In one embodiment, the oncogenic BRAF to be inhibited in the combinationtherapy of the invention has a mutation selected from the groupconsisting of VAL600GLU (also named as VAL599GLU) (Davies et al. Nature.Jun. 27, 2002; 417(6892):949-54), ARG461ILE, ILE462SER, GLY463GLU, andLYS600GLU (Rajagopalan, H., et al. (Letter) Nature 418: 934, 2002),GLY465VAL and LEU596ARG (Naoki, K., et al., Cancer Res. 62: 7001-7003,2002), and GLY468ARG, GLY468ALA and ASP593GLY (Lee, J. W., et al., Brit.J Cancer 89: 1958-1960, 2003), the references of which are eachincorporated herein by reference in their entirety. In one embodiment,the inhibitor binds specifically to oncogenic BRAF polypeptide.

Any inhibitor of oncogenic BRAF (e.g. BRAFV600E) can be used in methodsof the invention. As used herein, the phrase “inhibitor of oncogenicBRAF” means a compound or agent that inhibits the biological activity ofa BRAF oncogene, mutated or aberrantly expressed wild type. MultipleBRAF inhibitors are well known to those of skill in the art. Forexample, BRAF has been a target of small-molecule therapies to treatcancer (See e.g. Halilovic E, and Solit D B, Therapeutic strategies forinhibiting oncogenic BRAF signaling. Curr Opin Pharmacol 2008; 8:419-26;McCubrey J A et al. Targeting the Raf/MEK/ERK pathway withsmall-molecule inhibitors. Curr Opin Investig Drugs 2008, 9:614-30; andMichaloglou et al., Peeper D S. BRAF(E600) in benign and malignant humantumors. Oncogene 2008; 27:877-95).

Raf inhibitors, such as BAY 43-9006 (sorafenib), are not selective forBRAF, with activity against multiple kinase targets (Wilhelm S M, et al.(2004) BAY 43-9006 exhibits broad spectrum oral antitumor activity andtargets the RAF/MEK/ERK pathway and receptor tyrosine kinases involvedin tumor progression and angiogenesis. Cancer Res; 64:7099-109). Othersmall-molecule BRAF inhibitors include RAF265, XL281, AZ628, GSK2118436,and GDC-0879: See ClinicalTrials.gov. A study to evaluate RAF265, anoral drug administered to subjects with locally advanced or metastaticmelanoma. ClinicalTrials.gov identifier NCT00304525. Accessed Jan. 21,2010; Schwartz et al. (2008). A phase I study of XL281, a potent andselective inhibitor of RAF kinases, administrated orally to patientswith advanced solid tumors. 20th Eur J Cancer 6: 120, Abstract 383; andClinicalTrials.gov. A phase I study to investigate the safety,pharmacokinetics, and pharmacodynamics of GSK2118436 in subjects withsolid tumors. ClinicalTrials.gov identifier NCT00880321. Accessed Jan.21, 2010; Montagut et al (2008) Elevated CRAF as a potential mechanismof acquired resistance to BRAF inhibition in melanoma Cancer Res68:4853-61; and Hoeflich K P, et al (2009) Antitumor efficacy of thenovel RAF inhibitor GDC-0879 is predicted by BRAFV600E mutational statusand sustained extracellular signal-regulated kinase/mitogen-activatedprotein kinase pathway suppression. Cancer Res; 69: 3042-51.

BRAF inhibitors that have undergone clinical trials are described inShepard et al. (2010) B-RAF Inhibitors: An Evolving Role in the Therapyof Malignant Melanoma Current Oncology Reports Volume 12, Number 3,which is herein incorporated by reference in its entirety.

RG7204 (formerly PLX4032) is a small-molecule inhibitor that inhibitsBRAFV600E with a IC50 of 30 nmol/L (Sala E, et al. (2008) BRAF silencingby short hairpin RNA or chemical blockade by PLX4032 leads to differentresponses in melanoma and thyroid carcinoma cells. Mol Cancer Res 6:751-9).

In one embodiment, the inhibitor of oncogenic BRAF(V600E) is Vemurafenib(also known as PLX4032, RG7204 or RO5185426, and marketed as Zelboraftm(Hoffman-La Roches (Madisin, Wis.)/Daiichi Sankyo (Parsippany, N.J.)).Vemurafenib is a selective inhibitor of BRAF(V600E), and is described inU.S. Pat. No. 7,863,288, herein incorporated by reference in itsentirety.

In one embodiment, the inhibitor is a specific inhibitor of oncogenicBRAF(V600E) that is PLX4720 (Plexxikon Inc., Berkeley, Calif., USA)(Tsai J, Lee J T, Wang W, et al. Discovery of a selective inhibitor ofoncogenic B-Raf kinase with potent antimelanoma activity. Proc Natl AcadSci USA 2008; 105:3041-3046), see FIG. 5.

In addition, compounds that inhibit oncogenic BRAF (BRAF or mutatedBRAF) expression or activity may be readily identified using screeningmethods well known to those of skill in the art (e.g. see US2008/0072337). In one embodiment, compounds identified by the screeningmethods bind specifically to a BRAF nucleic acid or to BRAF polypeptide.In one embodiment, the compounds or agents antagonize BRAF and inhibit adownstream biological effect (e.g., inhibit the phosphorylation of MEKand ERK) that is associated with constitutive BRAF activity. In vivo orcell culture assays may be used to determine whether a test compoundfunctions as an antagonist to inhibit BRAF in cells. For instance, cellculture assays may be used to measure a test compound's ability tomodulate an activity, such as detecting inhibition of endogenousphospho-MEK levels, or increase sensitivity to chemotherapy, in tumorcells treated with a test compound. Such assays generally comprisecontacting a cell that expresses BRAF or mutated BRAF with a testcompound and comparing it to control cells not contacted with the testcompound. Cell assays include those utilizing conventional, reportergene-based assays, among others.

In one embodiment, the inhibitor of oncogenic BRAF is selected from thegroup consisting of Sorafenib (Bayer), RAF265 (Novartis), XL281(BMS-908662, Bristol-Myers Squibb; Exelixis), AZ628 (Montagut et al(2008) Cancer Res 68:4853-61), GSK2118436 (GlaxoSmithKline,), GDC-0879(Selleck Chemicals LLC, Houston, Tex.), PLX4032 (Vemurafenib, Plexxikon,Berkeley, Calif., USA), and PLX4720 (Difluorophenyl-sulfonamine,Plexxikon Inc., Berkeley, Calif., USA); and the inhibitor of DHODH isselected from the group consisting of: leflunomide, teriflunomide,brequinar, dichloroallyl lawsone, maritimus, redoxal and NSC210627.

In one specific embodiment the combination therapy described hereincomprises administration of an inhibitor of BRAF(V006E) that is PLX4720and an inhibitor of DHODH that is leflunomide or teriflunomide. Inanother specific embodiment, the combination therapy described hereincomprises administration of an inhibitor of BRAF(V006E) that is PLX4032and an inhibitor of DHODH that is leflunomide or teriflunomide.

In another embodiment, the invention contemplates the practice of themethod in conjunction with other therapies such as conventionalchemotherapy, radiation therapy or surgery directed against solid tumorsand for control of establishment of metastases. The administration ofangiogenesis-inhibiting amounts of combination therapy may be conductedbefore, during or after chemotherapy, radiation therapy or surgery.

Pharmaceutical Compositions and Administration

Embodiments of the invention comprise administering to a subject aninhibitor of oncogenic BRAF and/or and inhibitor of DHODH for thetreatment of melanoma. The administration of the DHODH inhibitor, orcombination therapy comprising administration of a DHODH and a BRAFinhibitor, may be for either “prophylactic” or “therapeutic” purpose.When provided prophylactically, therapy is provided in advance of anysymptom. The prophylactic administration of the therapy serves toprevent formation of melanoma. Prophylactic administration may be givento a patient with, for example, a family history of cancer, or a patientthat has had a melanoma removed surgically. Alternatively,administration of the combination therapy may be given to a patient withrising cancer marker protein levels, for example melanoma markersdescribed in PCT Publications WO 2008/141275, WO 2009/073513, or in U.S.Pat. No. 7,442,507

When provided therapeutically, the administration of the DHODHinhibitor, or combination therapy comprising administration of a DHODHinhibitor and a BRAF inhibitor, is provided at (or after) the onset of asymptom, or indication of melanoma.

For the combination therapy, the inhibitor of DHODH and the inhibitor ofBRAF can be present in the same or different pharmaceutical composition.When administrated at different times, the inhibitor of DHODH and theinhibitor of BRAF can be administered within 5 minutes, 10 minutes, 20minutes, 60 minutes, 2 hours, 3 hours, 4, hours, 8 hours, 12 hours, 24hours of administration of the other. When the inhibitors areadministered in different pharmaceutical compositions, routes ofadministration can be different.

The effective dosage range for the administration of the inhibitorsdepends upon the form of the inhibitor and its potency. It is an amountlarge enough to produce the desired effect in which symptoms of melanomaare ameliorated (e.g. inhibition of tumor growth). The phrase“therapeutically-effective amount” as used herein means that amount ofinhibitory compound or composition comprising the inhibitor/s which iseffective for producing the desired therapeutic effect, in at least asub-population of cells, in a subject at a reasonable benefit/risk ratioapplicable to any medical treatment. For example, an amount of acompound administered to a subject that is sufficient to produce astatistically significant, measurable change in at least one symptom ofmelanoma. Determination of a therapeutically effective amount is wellwithin the capability of those skilled in the art. Generally, atherapeutically effective amount can vary with the subject's history,age, condition, sex, as well as the severity and type of the medicalcondition in the subject, and administration of other pharmaceuticallyactive agents. There are preclinical melanoma models that are well knownto those of skill in the art which can be used to determinetherapeutically effective amounts of the compound or agents and tooptimize administration regimes. See for example Yang et al. (2010)RG7204 (PLX4032), A selective BRAFV600E inhibitor, displays potentantitumor activity in preclinical melanoma models, Cancer Research,70:5518-5527, which is herein incorporated by reference its entirety.

In one embodiment, a therapeutically effective amount of oncogenic BRAFinhibitor (e,g BRAF(V600E)) and/or DHODH inhibitor, inhibits melanomatumor volume in a preclinical model by at least 5%, at least 10%, atleast 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90% and reduces at least one symptomof melanoma by at least 5%, at least 10%, at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, or atleast 90%. For example, tumor volumes in xenograft mice can becalculated using the following ellipsoid formula: [D×(d2)]/2, in which Drepresents the large diameter of the tumor, and d represents the smalldiameter. Tumor volumes of treated groups are presented as percentagesof tumor volumes of the control groups (% T/C) using the followingformula: 100×[(T−T₀)/(C−C₀)], in which T represents mean tumor volume ofa treated group on a specific day during the experiment, T₀ representsmean tumor volume of the same treated group on the first day oftreatment, C represents mean tumor volume of a control group on thespecific day during the experiment, and C₀ represents mean tumor volumeof the same treated group on the first day of treatment. Percent tumorgrowth inhibition can be calculated as 100−% T/C, with >100% tumorgrowth inhibition representing regression. Survival can be calculatedusing a predefined cutoff volume of 2,000 mm³ as a surrogate formortality (See e.g. Yang et al. (2010), Supra).

In one embodiment a therapeutically effective amount of the DHODHinhibitor inhibits the enzymatic activity of DHODH by at least 5%, atleast 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, or at least 90% and reduces atleast one symptom of melanoma by at least 5%, at least 10%, at least20%, at least 30%, at least 40%, at least 50%, at least 60%, at least70%, at least 80%, or at least 90%.

In one embodiment a therapeutically effective amount of the oncogenicBRAF inhibitor and/or DHODH inhibitor inhibits cellular proliferation ina preclinical model by at least 5%, at least 10%, at least 20%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least80%, or at least 90% and reduces at least one symptom of melanoma by atleast 5%, at least 10%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, or at least 90%Inhibition of cellular proliferation may be evaluated by3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT;Sigma) assay. For example cells can be plated in 96-well microtiterplates at a density of 1,000 to 5,000 cells per well in a volume of 180μL. Twenty-four hours after cell plating, 20 μL of an appropriatecompound/agent dilution can be added to plates in duplicate. The platesmay then be assayed for proliferation 6 days after the cells were platedaccording to the procedure originally described by Mosmann, Rapidcolomeric assay for cellular growth and survival: application toproliferation and cytotoxicity assays. J. Immunol. Methods1883:65:55-63). Percent inhibition can then be calculated and the IC50determined from the regression of a plot of the logarithm of theconcentration versus percent inhibition by XLfit (version 4.2; IDBS)using a Dose-Response One-Site Model (#205) (see e.g. Yang et al. Supra)

The therapeutically effective dose can be estimated initially from asuitable cell culture or enzymatic assays (e.g. melanoma cell growthassays, phosphorylation assays, or DHODH enzymatic activity assays),then a dose of each compound and treatment regime may be formulated inanimal models to achieve a circulating plasma concentration range thatincludes the IC50 as determined in cell culture.

For administration to a subject, the compounds or agents can be providedin pharmaceutically acceptable compositions. These pharmaceuticallyacceptable compositions comprise a therapeutically-effective amount ofone or more of inhibitors, formulated together with one or morepharmaceutically acceptable carriers (additives) and/or diluents. Thepharmaceutical compositions of the present invention can be speciallyformulated for administration in solid or liquid form, including thoseadapted for the following: (1) oral administration, for example,drenches (aqueous or non-aqueous solutions or suspensions), lozenges,dragees, capsules, pills, tablets (e.g., those targeted for buccal,sublingual, and systemic absorption), boluses, powders, granules, pastesfor application to the tongue; (2) parenteral administration, forexample, by subcutaneous, intramuscular, intravenous or epiduralinjection as, for example, a sterile solution or suspension, orsustained-release formulation; (3) topical application, for example, asa cream, ointment, or a controlled-release patch or spray applied to theskin; (4) intravaginally or intrarectally, for example, as a pessary,cream or foam; (5) sublingually; (6) ocularly; (7) transdermally; (8)transmucosally (e.g. as a nasal spray or suppository); or (9) nasally.Additionally, compounds can be implanted into a patient or injectedusing a drug delivery system. See, for example, Urquhart, et al., Ann.Rev. Pharmacol. Toxicol. 24: 199-236 (1984); Lewis, ed. “ControlledRelease of Pesticides and Pharmaceuticals” (Plenum Press, New York,1981); U.S. Pat. No. 3,773,919; and U.S. Pat. No. 35 3,270,960. Guidancefor formulations can be found in e.g. Remington: The Science andPractice of Pharmacy by Alfonso R. Gelmaro (Ed.) 20^(th) edition: Dec.15, 2000, Lippincott, Williams $ Wilkins, ISBN: 0683306472, and arebriefly described below.

As used here, the term “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

As used here, the term “pharmaceutically-acceptable carrier” means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, manufacturing aid (e.g.,lubricant, talc magnesium, calcium or zinc stearate, or steric acid), orsolvent encapsulating material, involved in carrying or transporting thesubject compound from one organ, or portion of the body, to anotherorgan, or portion of the body. Each carrier must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not injurious to the patient. Some examples of materials which canserve as pharmaceutically-acceptable carriers include: (1) sugars, suchas lactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, methylcellulose, ethyl cellulose,microcrystalline cellulose and cellulose acetate; (4) powderedtragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such asmagnesium stearate, sodium lauryl sulfate and talc; (8) excipients, suchas cocoa butter and suppository waxes; (9) oils, such as peanut oil,cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12)esters, such as ethyl oleate and ethyl laurate; (13) agar; (14)buffering agents, such as magnesium hydroxide and aluminum hydroxide;(15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18)Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents,such as polypeptides and amino acids (23) serum component, such as serumalbumin, HDL and LDL; (22) C₂-C₁₂ alcohols, such as ethanol; and (23)other non-toxic compatible substances employed in pharmaceuticalformulations. Wetting agents, coloring agents, release agents, coatingagents, sweetening agents, flavoring agents, perfuming agents,preservative and antioxidants can also be present in the formulation.The amount of compound which can be combined with a carrier material toproduce a single dosage form will generally be that amount of thecompound which produces a therapeutic effect. Generally out of onehundred percent, this amount will range from about 0.1% to 99% ofcompound, preferably from about 5% to about 70%, most preferably from10% to about 30%.

Formulations suitable for parenteral administration conveniently includesterile aqueous preparation of the active compound which is preferablyisotonic with the blood of the recipient. Thus, such formulations mayconveniently contain distilled water, 5% dextrose in distilled water orsaline. Useful formulations also include concentrated solutions orsolids containing the compound which upon dilution with an appropriatesolvent give a solution suitable for parental administration above.

For enteral administration, a compound can be incorporated into an inertcarrier in discrete units such as capsules, cachets, tablets orlozenges, each containing a predetermined amount of the active compound;as a powder or granules; or a suspension or solution in an aqueousliquid or non-aqueous liquid, e.g., a syrup, an elixir, an emulsion or adraught. Suitable carriers may be starches or sugars and includelubricants, flavorings, binders, and other materials of the same nature.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active compound in a free-flowingform, e.g., a powder or granules, optionally mixed with accessoryingredients, e.g., binders, lubricants, inert diluents, surface activeor dispersing agents. Molded tablets may be made by molding in asuitable machine, a mixture of the powdered active compound with anysuitable carrier.

A syrup or suspension may be made by adding the active compound to aconcentrated, aqueous solution of a sugar, e.g., sucrose, to which mayalso be added any accessory ingredients. Such accessory ingredients mayinclude flavoring, an agent to retard crystallization of the sugar or anagent to increase the solubility of any other ingredient, e.g., as apolyhydric alcohol, for example, glycerol or sorbitol.

Formulations for rectal administration may be presented as a suppositorywith a conventional carrier, e.g., cocoa butter or Witepsol S55(trademark of Dynamite Nobel Chemical, Germany), for a suppository base.

Formulations for oral administration may be presented with an enhancer.Orally-acceptable absorption enhancers include surfactants such assodium lauryl sulfate, palmitoyl carnitine, Laureth-9,phosphatidylcholine, cyclodextrin and derivatives thereof; bile saltssuch as sodium deoxycholate, sodium taurocholate, sodium glycocholate,and sodium fusidate; chelating agents including EDTA, citric acid andsalicylates; and fatty acids (e.g., oleic acid, lauric acid,acylcarnitines, mono- and diglycerides). Other oral absorption enhancersinclude benzalkonium chloride, benzethonium chloride, CHAPS(3-(3-cholamidopropyl)-dimethylammonio-1-propanesulfonate), Big-CHAPS(N, N-bis(3-D-gluconamidopropyl)-cholamide), chlorobutanol, octoxynol-9,benzyl alcohol, phenols, cresols, and alkyl alcohols. An especiallypreferred oral absorption enhancer for the present invention is sodiumlauryl sulfate.

As used herein, the term “administer” or “administering” refers to theplacement of a composition into a subject by a method or route whichresults in at least partial localization of the composition at a desiredsite such that desired effect is produced. A compound or compositiondescribed herein can be administered by any appropriate route known inthe art including, but not limited to, oral or parenteral routes,including intravenous, intramuscular, subcutaneous, transdermal, airway(aerosol), pulmonary, nasal, rectal, and topical (including buccal andsublingual) administration.

Exemplary modes of administration include, but are not limited to,injection, infusion, instillation, inhalation, or ingestion. “Injection”includes, without limitation, intravenous, intramuscular, intraarterial,intrathecal, intraventricular, intracapsular, intraorbital,intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous,subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal,intracerebro spinal, and intrasternal injection and infusion.

Methods of delivering RNAi interfering (RNAi) agents (e.g., an siRNA),other nucleic acid modulators, or vectors containing modulatory nucleicacids, to the target cells (e.g., melanocytes) can include, for exampledirectly contacting the cell with a composition comprising a modulatorynucleic acid, or local or systemic injection of a composition containingthe modulatory nucleic acid. In one embodiment, nucleic acid agents(e.g. RNAi, siRNA, or other nucleic acid) are injected directly into atumor. In some embodiments modulatory nucleic may be delivered bysystemic administration, wherein the nucleic acid is complexed with, oralternatively contained within a carrier. Example carriers formodulatory nucleic acid compounds include, but are not limited to,peptide carriers, viral vectors, gene therapy reagents, and/or liposomecarrier complexes and the like.

Alternatively, the compound may be administered in liposomes ormicrospheres (or microparticles). Methods for preparing liposomes andmicrospheres for administration to a patient are well known to those ofskill in the art. U.S. Pat. No. 4,789,734, the contents of which arehereby incorporated by reference, describes methods for encapsulatingbiological materials in liposomes. A review of known methods is providedby G. Gregoriadis, Chapter 14, “Liposomes,” Drug Carriers in Biology andMedicine, pp. 287-341 (Academic Press, 1979) also U.S. Pat. Nos.4,906,474, 4,925,673 and 3,625,214, and Jein, TIPS 19:155-157 (1998),the contents of which are hereby incorporated by reference.

In some embodiments, the compound/agents described herein for treatmentof melanoma can be administered to a subject in combination withadditional pharmaceutically active agents. Exemplary pharmaceuticallyactive compound include, but are not limited to, those found inHarrison's Principles of Internal Medicine, 13^(th) Edition, Eds. T. R.Harrison et al. McGraw-Hill N.Y., NY; Physicians Desk Reference, 50^(th)Edition, 1997, Oradell N.J., Medical Economics Co.; PharmacologicalBasis of Therapeutics, 8^(th) Edition, Goodman and Gilman, 1990; UnitedStates Pharmacopeia, The National Formulary, USP XII NF XVII, 1990;current edition of Goodman and Oilman's The Pharmacological Basis ofTherapeutics; and current edition of The Merck Index, the completecontents of all of which are incorporated herein by reference.

Toxicity and therapeutic efficacy can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compositions that exhibit large therapeutic indices, are preferred.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized.

The therapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range that includes the IC50 (i.e., theconcentration of the therapeutic which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Levels in plasmamay be measured, for example, by high performance liquid chromatography.The effects of any particular dosage can be monitored by a suitablebioassay.

The dosage may be determined by a physician and adjusted, as necessary,to suit observed effects of the treatment. Generally, the compositionsare administered so that a compound is given at a dose from 1 μg/kg to150 mg/kg, 1 μg/kg to 100 mg/kg, 1 μg/kg to 50 mg/kg, 1 μg/kg to 20mg/kg, 1 μg/kg to 10 mg/kg, 1 μg/kg to 1 mg/kg, 100 μg/kg to 100 mg/kg,100 μg/kg to 50 mg/kg, 100 μg/kg to 20 mg/kg, 100 μg/kg to 10 mg/kg, 100μg/kg to 1 mg/kg, 1 mg/kg to 100 mg/kg, 1 mg/kg to 50 mg/kg, 1 mg/kg to20 mg/kg, 1 mg/kg to 10 mg/kg, 10 mg/kg to 100 mg/kg, 10 mg/kg to 50mg/kg, or 10 mg/kg to 20 mg/kg. It is to be understood that ranges givenhere include all intermediate ranges, for example, the range 1 mg/kg to10 mg/kg includes 1 mg/kg to 2 mg/kg, 1 mg/kg to 3 mg/kg, 1 mg/kg to 4mg/kg, 1 mg/kg to 5 mg/kg, 1 mg/kg to 6 mg/kg, 1 mg/kg to 7 mg/kg, 1mg/kg to 8 mg/kg, 1 mg/kg to 9 mg/kg, 2 mg/kg to 10 mg/kg, 3 mg/kg to 10mg/kg, 4 mg/kg to 10 mg/kg, 5 mg/kg to 10 mg/kg, 6 mg/kg to 10 mg/kg, 7mg/kg to 10 mg/kg, 8 mg/kg to 10 mg/kg, 9 mg/kg to 10 mg/kg etc. . . . .It is to be further understood that the ranges intermediate to the givenabove are also within the scope of this invention, for example, in therange 1 mg/kg to 10 mg/kg, dose ranges such as 2 mg/kg to 8 mg/kg, 3mg/kg to 7 mg/kg, 4 mg/kg to 6 mg/kg etc.

With respect to duration and frequency of treatment, it is typical forskilled clinicians to monitor subjects in order to determine when thetreatment is providing therapeutic benefit, and to determine whether toincrease or decrease dosage, increase or decrease administrationfrequency, discontinue treatment, resume treatment or make otheralteration to treatment regimen. The dosing schedule can vary from oncea week to daily depending on a number of clinical factors, such as thesubject's sensitivity.

The desired dose can be administered at one time or divided intosubdoses, e.g., 2-4 subdoses and administered over a period of time,e.g., at appropriate intervals through the day or other appropriateschedule. Such sub-doses can be administered as unit dosage forms. Insome embodiments, administration is chronic, e.g., one or more dosesdaily over a period of weeks or months. Examples of dosing schedules areadministration daily, twice daily, three times daily or four or moretimes daily over a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month,2 months, 3 months, 4 months, 5 months, or 6 months or more. Thepharmaceutical compositions can be administered during infancy (between0 to about 1 year of life), childhood (the period of life betweeninfancy and puberty) and during puberty (between about 8 years of lifeto 18 years of life). The pharmaceutical compositions can also beadministered to treat adults (greater than about 18 years of life). Adose administered at least once, may be provided as a bolus, acontinuous administration or sustained release. Multiple administrationover a period of weeks or months may be preferable. It may also bepreferable to administer the dose at least once/week and even morefrequent administrations (e.g. daily). Subsequent doses may beadministered as indicated.

In one embodiment, the inhibitor of DHODH (e.g. leflunomide) isadministered to a subject using an administration regime that results ina steady state concentration of 70 mcg/mL, or 60 mcg/mL, or 50 mcg/mL,40 mcg/mL, 30 mcg/ml, or 20 mcg/mL (Rozman, B. (2002) Clinicalpharmacokinetics of leflunomide, Clin. Pharmacokinetic, 41:421-430). Inone embodiment, the steady state concentration is 60 mcg/mL or less, 50mcg/mL or less, 40 mcg/mL or less, 30 mcg/ml or less, or 20 mcg/mL orless.

In one embodiment, the inhibitor of DHODH (e.g. leflunomide) isadministered at 100 mg daily for 3 days, followed by lower daily dosesof 20 mg/ml for a sustained concentration of 60 mcg/mL.

In one embodiment, the inhibitor of BRAF (e.g. PLX4302) is administeredat a dosage of 960 mg twice a day. In one embodiment, the inhibitor ofBRAF (e.g. PLX4302) is administered at a dosage less than 900 mg twice aday, less than 850 mg twice a day, less than 800 mg twice a day, or lessthan 700 mg twice per day.

The efficacy of a given treatment regime for melanoma can be determinedby the skilled clinician, for example by assessing physical indicatorsof melanoma, such as e.g., tumor size or lesion size, metastasis, tumorgrowth rate, etc. However, a treatment is considered “effectivetreatment,” as the term is used herein, if any one or all of the signsor symptoms of the cancer are altered in a beneficial manner, e.g.improved or ameliorated by at least 10% following treatment with acompound or agent that inhibits BRAF and/or DHODH. Efficacy can also bemeasured by a failure of an individual to worsen as assessed bystabilization of tumor growth, hospitalization or need for medicalinterventions (i.e., progression of the melanoma is halted or at leastslowed). Methods of measuring these indicators are known to those ofskill in the art and/or described herein. Treatment includes in anindividual includes: (1) inhibiting the disease, e.g., arresting, orslowing melanoma tumor or lesion growth; or (2) relieving the disease,e.g., causing regression of symptoms, reducing tumor or lesion size; and(3) preventing or reducing the likelihood of the development ofmelanoma, or preventing metastasis of the melanoma.

Screening Methods

In another aspect of the invention, methods of screening for compoundsthat are useful for treatment of melanoma are provided.

We have previously described a transgenic zebrafish melanoma model(Patton, E. E. et al. BRAF mutations are sufficient to promote neviformation and cooperate with p53 in the genesis of melanoma. Curr. Biol15, 249-54 (2005); U.S. 2008/0072337). Transgenic zebrafish expressinghuman BRAF(V600E) under the melanocyte-specific mitf promoter(mitf-BRAF(V600E)) develop melanoma at 4-12 months of age when crossedwith p53−/− mutants. Herein, we determined, by gene expression analysis,that the melanoma cells adopted a fate similar to that of the BRAFV600E;p53−/− embryos (See Examples). We discovered that BRAF(V600E):p53−/−embryos exhibit an abnormal expansion in the number of crestin+progenitors (marker of embryonic neural crest progenitors), and that inthe adult BRAF(V600E):p53−/− virtually all tumor cells, but no normalcells, are positive for crestin. Crestin is normally downregulated afterterminal differentiation of neural crest progenitors. Thus, we developeda screen for compounds useful for melanoma treatment that comprisesscreening for inhibitors of neural crest progenitors duringembryogenesis, e.g. screening for inhibitors of the crestin+ lineage.

Methods of screening for agents that inhibit melanoma growth areprovided. The method comprises: (a) contacting a zebrafish embryo with atest agent for a period of time (e.g. hours, days, or weeks), (b)rinsing the embryos of step (a); and (c) assaying the number of neuralcrest progenitors as compared to a control zebrafish embryo that has notbeen contacted with the test agent, wherein a reduced number of neuralcrest progenitors indicates that the compound is capable of inhibitingmelanoma growth.

In one embodiment, the method further comprises the step of measuringinhibition of melanoma growth, e.g. in a transgenic melanoma zebrafishmodel, or in a melanoma proliferation assay, or mouse xenograft melanomamodel (See e.g. Examples and U.S. patent application publication2008/0072337).

The embryos may be contacted at any time of embryonic development. Inone embodiment, the embryos are contacted with an agent at approximately20%, 30%, 40%, 50%, 60%, or 70% epiboly.

In one embodiment, the zebrafish embryos used in the screen are wildtype (wt) zebrafish embryos.

In one embodiment, inhibition of neural crest progenitors is scored bymonitoring for inhibition of crestin expression in cells of the neuralcrest of the embryo. Crestin expression can be monitored by any meansknown to those of skill in the art, including but not limited to in situhybridization with a crestin riboprobe. When monitoring expression ofcrestin by ISH the method further comprises the step of fixing theembryos prior to performing ISH using means well known to those of skillin the art (e.g. fixing in PFA and methanol). Expression can be scoredusing visualization, or other means known to those of skill in the art,e.g. manual scoring may comprises assigning a score within a range of 1(no crestin staining) to 3 (no change in crestin) to 5, a markedincrease in crestin, e.g. compounds having a score of 2 or less may beconsidered to inhibit the number of crestin⁺ cells.

In one embodiment the zebrafish embryos are of mitfa-GFP transgeniczebrafish (express green fluorescent protein (GFP) operably linked tomitfa melanocyte specific promoter), and inhibition of neural crestprogenitors is scored by monitoring for inhibition of the number ofmelanocytes along the neural crest of the embryo, e.g. by determiningthe amount of GFP expression in the neural crest, e.g. by visual scoringor computerized means.

In one embodiment, inhibition of neural crest progenitors is scored bymonitoring for inhibition of sox10 expression, or dct expression incells of the neural crest of the embryo.

The methods of screening described herein, can be performed as highthroughput assays. For example a plurality of embryos may be contactedwith a plurality of compounds or agents, e.g. in a multi-well format,and the step of assaying the number of neural crest progenitors may beatomized, e.g. using automated in situ hybridization or fluorescenceimaging. Compounds showing inhibition of neural crest progenitors maythen be selected.

An exemplary assay may involve plating wild type zebrafish embryos inmulti-well plates in appropriate media, e.g. at a density of about 10embryos per well, and at about 50% epiboly, adding the test agent to thewells. Any concentration of test agent can be used. In one embodiment,the test agent concentration ranges from, 1 uM to 50 uM, 0.1 nm to 1000mM, or e.g. 1 uM to 1 mm, or more. High concentrations may be toxic tothe embryos, which can easily be determined by testing and theconcentration can be adjusted to minimize toxicity. 24 hours aftertreatment the cells embryos are rinsed of test drug and fixed for insitu hybridization, e.g. of crestin, sox10, dct, edn, etc. Compoundsresulting in statistically significant inhibition of neural crestprogenitor cells are selected, and are optionally tested forquantitation of inhibition of melanoma growth in other assays, e.g. %inhibition of melanocyte proliferation (See Examples), or tumorinhibition studies, e.g. using xenograft mice. Means for plating andmanipulating zebrafish are well known to those of skill in the art, seee.g. Kaufman, C. K., white, R. M. & Zon, L. chemical genetic screeningin zebrafish embryo. Nat. Protoc. 4, 1422-1432 (2009). This is merely anexemplary assay, there are multiple means well known to those of skillin the art to monitor neural crest progenitor cell formation that aresuitable for use in the screening assay.

In an alternative embodiment, the zebrafish embryo used in the screeningmethods described herein, has been genetically manipulated to express anucleic acid encoding a mutant human BRAF and a mutant tumor suppressorand/or protooncogene, e.g. mitf-BRAF(V600E); p53−/−, or other oncogenicBRAF zebrafish melanoma models. Such models are described in detail inU.S. patent application publication 2008/0072337, which is hereinincorporated by reference in its entirety.

As used herein, the term “test compound” refers to compounds and/orcompositions that are to be screened for their ability to inhibit thenumber of neural crest progenitors. The test compounds can include awide variety of different compounds, including chemical compounds andmixtures of chemical compounds, e.g., small organic or inorganicmolecules; saccharines; oligosaccharides; polysaccharides; biologicalmacromolecules, e.g., peptides, proteins, and peptide analogs andderivatives; peptidomimetics; nucleic acids; nucleic acid analogs andderivatives; an extract made from biological materials such as bacteria,plants, fungi, or animal cells; animal tissues; naturally occurring orsynthetic compositions; and any combinations thereof. In someembodiments, the test compound is a small molecule.

As used herein, the term “small molecule” refers to in organic ororganic compounds. However, small molecules typically are characterizedin that they contain several carbon-carbon bonds, and have a molecularweight of less than 5000 Daltons (5 kD), preferably less than 3 kD,still more preferably less than 2 kD, and most preferably less than 1kD. In some cases it is preferred that a small molecule has a molecularweight equal to or less than 700 Daltons.

The number of possible test compounds runs into millions. Methods fordeveloping small molecule, polymeric and genome based libraries aredescribed, for example, in Ding, et al. J Am. Chem. Soc. 124: 1594-1596(2002) and Lynn, et al., J. Am. Chem. Soc. 123: 8155-8156 (2001).Commercially available compound libraries can be obtained from, e.g.,ArQule, Pharmacopia, graffinity, Panvera, Vitas-M Lab, BiomolInternational and Oxford. These libraries can be screened using thescreening devices and methods described herein. Chemical compoundlibraries such as those from NIH Roadmap, Molecular Libraries ScreeningCenters Network (MLSCN) can also be used. Compound libraries are wellknown and readily available in the art. A chemical library or compoundlibrary is a collection of stored chemicals usually used ultimately inhigh-throughput screening or industrial manufacture. The chemicallibrary can consist in simple terms of a series of stored chemicals.Each chemical has associated information stored in some kind of databasewith information such as the chemical structure, purity, quantity, andphysiochemical characteristics of the compound.

In the methods of the invention, the compounds/agents are typicallyprovided free in solution, however the compounds/or agent may be incomplex with solid forms.

In some embodiments, the test compound inhibits neural crest progenitorsby at least 5%, 10%, 20%, 30%, 40%, 50%, 50%, 70%, 80%, 90%, 1-fold,1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 50-fold,100-fold or more higher relative to an untreated control.

Definitions

As used herein the term “comprising” or “comprises” is used in referenceto compositions, methods, and respective component(s) thereof, that areessential to the invention, yet open to the inclusion of unspecifiedelements, whether essential or not.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof additional elements that do not materially affect the basic and novelor functional characteristic(s) of that embodiment of the invention.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages maymean±1%.

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. It is further to be understood that all base sizes or aminoacid sizes, and all molecular weight or molecular mass values, given fornucleic acids or polypeptides are approximate, and are provided fordescription.

The abbreviation, “e.g.” is derived from the Latin exempli gratia, andis used herein to indicate a non-limiting example. Thus, theabbreviation “e.g.” is synonymous with the term “for example.”

The terms “decrease”, “reduced”, “reduction”, “decrease” or “inhibit”are all used herein generally to mean a decrease by a statisticallysignificant amount. However, for avoidance of doubt, “reduced”,“reduction” or “decrease” or “inhibit” means a decrease by at least 10%as compared to a reference level, for example a decrease by at leastabout 20%, or at least about 30%, or at least about 40%, or at leastabout 50%, or at least about 60%, or at least about 70%, or at leastabout 80%, or at least about 90% or up to and including a 100% decrease(e.g. absent level as compared to a reference sample), or any decreasebetween 10-100% as compared to a reference level.

The terms “increased”, “increase” or “enhance” or “activate” are allused herein to generally mean an increase by a statically significantamount; for the avoidance of any doubt, the terms “increased”,“increase” or “enhance” or “activate” means an increase of at least 10%as compared to a reference level, for example an increase of at leastabout 20%, or at least about 30%, or at least about 40%, or at leastabout 50%, or at least about 60%, or at least about 70%, or at leastabout 80%, or at least about 90% or up to and including a 100% increaseor any increase between 10-100% as compared to a reference level, or atleast about a 2-fold, or at least about a 3-fold, or at least about a4-fold, or at least about a 5-fold or at least about a 10-fold increase,or any increase between 2-fold and 10-fold or greater as compared to areference level.

The term “statistically significant” or “significantly” refers tostatistical significance and generally means a two standard deviation(2SD) below normal, or lower, concentration of the marker. The termrefers to statistical evidence that there is a difference. It is definedas the probability of making a decision to reject the null hypothesiswhen the null hypothesis is actually true. The decision is often madeusing the p-value.

As used herein, the term “IC50” refers to the concentration of aninhibitor that produces 50% of the maximal inhibition of activity orexpression measurable using the same assay in the absence of theinhibitor. The IC50 can be as measured in vitro or in vivo. The IC50 canbe determined by measuring activity using a conventional in vitro assay(e.g. protein activity assay, or gene expression assay).

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such may vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention, which is defined solely by the claims.

As used herein and in the claims, the singular forms include the pluralreference and vice versa unless the context clearly indicates otherwise.Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.”

All patents and other publications identified are expressly incorporatedherein by reference for the purpose of describing and disclosing, forexample, the methodologies described in such publications that might beused in connection with the present invention. These publications areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing in this regard should be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention or for any other reason. Allstatements as to the date or representation as to the contents of thesedocuments is based on the information available to the applicants anddoes not constitute any admission as to the correctness of the dates orcontents of these documents.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as those commonly understood to one of ordinaryskill in the art to which this invention pertains.

The present invention may be as defined in any one of the followingnumbered paragraphs.

1. A method for treating melanoma in a subject comprising administeringto a subject in need thereof a therapeutically effective amount of aninhibitor of dihydroorotate dehydrogenase (DHODH).

2. The method of para. 1, wherein the inhibitor of dihydroorotatedehydrogenase (DHODH) is selected from the group consisting of:leflunomide, teriflunomide, brequinar, dichloroallyl lawsone, maritimus,redoxal and NSC210627, or a derivative thereof.

3. A method for treating melanoma in a subject comprising administeringto a subject in need thereof a therapeutically effective amount of aninhibitor of dihydroorotate dehydrogenase (DHODH) and an effectiveamount of an inhibitor of oncogenic BRAF.

4. The method of para. 3, wherein the inhibitor of oncogenic BRAF isselected from the group consisting of: a small molecule, a nucleic acidRNA, a nucleic acid DNA, a protein, a peptide, and an antibody.

5. The method of any of para.'s 1-4, wherein the inhibitor ofdihydroorotate dehydrogenase is selected from the group consisting of: asmall molecule, a nucleic acid RNA, a nucleic acid DNA, a protein, apeptide, and an antibody.

6. The method of any of para.'s 3-5, wherein the oncogenic BRAF isBRAF(V600E).

7. The method of any of para.'s 3-6, wherein the inhibitor of oncogenicBRAF is selected from the group consisting of: Sorafenib, RAF265, XL281,AZ628, GSK2118436, GDC-0879, PLX4032, and PLX4720.

8. The method of any of para.'s 3-5, wherein the inhibitor ofdihydroorotate dehydrogenase (DHODH) is selected from the groupconsisting of leflunomide, teriflunomide, brequinar, dichloroallyllawsone, maritimus, redoxal and NSC210627.

9. The method of any of para.'s 3-6, wherein the inhibitor of oncogenicBRAF is PLX4032 and the inhibitor of dihydroorotate dehydrogenase(DHODH) is leflunomide, or a derivative thereof.

10. The method of any of para.'s 3-6, wherein the inhibitor of oncogenicBRAF is PLX4720 and the inhibitor of dihydroorotate dehydrogenase(DHODH) is leflunomide, or a derivative thereof.

11. The method of any of para.'s 3-10, wherein the inhibitor ofoncogenic BRAF and inhibitor of dihydroorotate dehydrogenase (DHODH) areadministered to the subject sequentially or simultaneously.

12. A method of screening for an agent that inhibits melanoma growthcomprising (a) contacting a zebrafish embryo with a test agent for aperiod of time,

-   -   (b) rinsing the test agent from the embryos of step (a); and    -   (c) assaying the number of neural crest progenitors as compared        to a control zebrafish embryo that has not been contacted with        the test agent,    -   wherein a reduced number of neural crest progenitors indicates        that the compound is capable of inhibiting melanoma.

13. The method of para. 12, wherein the reduced number of neuralprogenitors is due to their differentiation to melanocytes (e.g. ameasured using melanocyte markers or assessing terminal differentiation)

14. The method of paras. 12-13, wherein the zebrafish embryo is a wildtype zebra fish embryo.

15. The method of any of para.'s 12-14, wherein the zebrafish embryo isa transgenic zebra fish embryo.

16. The method of para. 15, wherein the transgenic zebrafish expressesgreen fluorescent protein operably linked to the melanocyte mitfapromoter.

17. The method of any of para.'s 1-16, wherein the number of neuralcrest progenitors is assayed by monitoring crestin expression.

18. The method of any of para.'s 1-17, wherein the number of neuralcrest progenitors is assayed by monitoring sox10 expression.

19. The method of any of para.'s 1-18, wherein the number of neuralcrest progenitors is assayed by monitoring dct expression.

20. Use of an effective amount of an inhibitor of dihydroorotatedehydrogenase (DHODH) for the manufacture of a medicament for treatmentof melanoma.

21. The use of para. 20, wherein the inhibitor of dihydroorotatedehydrogenase (DHODH) is selected from the group consisting of:leflunomide, teriflunomide, brequinar, dichloroallyl lawsone, maritimus,redoxal and NSC210627, or a derivative thereof.

22. Use of an effective amount of an inhibitor of dihydroorotatedehydrogenase (DHODH) and an effective amount of an inhibitor ofoncogenic BRAF for the treatment of melanoma.

23. The use of para. 22, wherein the inhibitor of oncogenic BRAF isselected from the group consisting of: a small molecule, a nucleic acidRNA, a nucleic acid DNA, a protein, a peptide, and an antibody.

24. The use of any of para.'s 19-23, wherein the inhibitor ofdihydroorotate dehydrogenase is selected from the group consisting of: asmall molecule, a nucleic acid RNA, a nucleic acid DNA, a protein, apeptide, and an antibody.

25. The use of any of the para.'s 21-24, wherein the oncogenic BRAF isBRAF(V600E).

26. The use of any of para.'s 21-25, wherein the inhibitor of oncogenicBRAF is selected from the group consisting of: Sorafenib, RAF265, XL281,AZ628, GSK2118436, GDC-0879, PLX4032, and PLX4720.

27. The use of any of para.'s 21-26, wherein the inhibitor ofdihydroorotate dehydrogenase (DHODH) is selected from the groupconsisting of leflunomide, teriflunomide, brequinar, dichloroallyllawsone, maritimus, redoxal and NSC210627.

28. The use of any of para.'s 21-27, wherein the inhibitor of oncogenicBRAF is PLX4032 and the inhibitor of dihydroorotate dehydrogenase(DHODH) is leflunomide, or a derivative thereof.

29. The use of any of para.'s 21-28, wherein the inhibitor of oncogenicBRAF is PLX4720 and the inhibitor of dihydroorotate dehydrogenase(DHODH) is leflunomide, or a derivative thereof.

30. The method of any of para.'s 21-29, wherein the inhibitor ofoncogenic BRAF and inhibitor of dihydroorotate dehydrogenase (DHODH) areadministered to the subject sequentially or simultaneously.

All references described herein, in the Examples and throughout theSpecification, are incorporated herein by reference in their entirety.

EXAMPLES Example I

In melanoma, it is unknown to what extent BRAF^(V600E) mutations dependupon transcriptional programs present in the developmental lineage oftumor initiation. These programs may be therapeutic targets whencombined with BRAF^(V600E) inhibition. We have utilized zebrafishembryos to identify small molecule suppressors of neural crestprogenitors which give rise to melanoma. Transgenic zebrafish expressinghuman BRAF^(V600E) under the melanocyte-specific mitf promoter(mitf-BRAF^(V600E)) develop melanoma at 4-12 months of age when crossedwith p53^(−/−) mutants (FIG. 1a ). As the mitf promoter drivesBRAF^(V600E) starting at 16 hours post fertilization (hpf), overlappingwith other markers such as sox10, events that occur early inembryogenesis are analogous to those occurring at tumor initiation. Togain insight into initiating events, we compared gene expressionprofiles of BRAF^(V600E);p53^(−/−) embryos to BRAF^(V600E);p53^(−/−)melanomas using Gene Set Enrichment Analysis (GSEA) (FIG. 1b ). Thisrevealed a 123 gene overlap signature, notable for markers of embryonicneural crest progenitors (crestin, sox10, ednrb) and melanocytes (tyr,dct) (data not shown). This is similar to that of a multipotent neuralcrest progenitor, and suggested that the melanoma adopted this fate.

We analyzed alterations in embryonic neural crest development using insitu hybridization (ISH). At 24 hpf, BRAF^(V600E);p53^(−/−) embryosexhibit an abnormal expansion in the number of crestin⁺ progenitors,along with an increase in other markers from the 123 gene signature suchas spry4 and rab3il1 and eden1 (data not shown). Hema toxylin & Eosin (H& E) and crestin ISH on zebrafish melanoma from the dorsum of the fish.Although only a small fraction of the tumor shows pigmented cells,virtually the entire tumor mass was positive for the neural crest markercrestin (data not shown). By 72 hpf, crestin aberrantly persists withinthe head, tail and dorsal epidermis only in BRAF^(V600E);p53^(−/−)embryos and is absent in non tumor borne strains (data not shown).crestin, a zebrafish specific gene², is normally downregulated afterterminal differentiation of neural crest progenitors³, suggesting thatactivated BRAF^(V600E) promotes maintenance of multipotency in neuralcrest progenitors, which become expanded during tumorigenesis. In adultBRAF^(V600E);p53^(−/−) melanomas, virtually all tumor cells, but nonormal cells, were positive for crestin (FIG. 1c ). Only 10-15% of themelanoma cells are pigmented (FIG. 9), consistent with the concept thatadult zebrafish melanomas retain a progenitor-like state. A humanmelanoma tissue array showed similar findings: 75.0% were positive forthe neural crest progenitor gene ednrb, but 12.8% for the melanocytelineage marker dct (FIG. 9), in agreement with findings that most humanmelanomas express the neural crest marker sox10⁴. These data indicatethat the majority of human melanomas reflect events that lead to themaintenance of a neural crest progenitor phenotype⁵.

We hypothesized that chemical suppressors of neural crest progenitorswould have utility in treatment of melanoma. We screened 2,000 chemicalsto identify inhibitors of the crestin⁺ lineage during embryogenesis.Most chemicals (90%) had minimal effect or were toxic (FIG. 10).NSC210627, a molecule of unknown function, strongly abrogated expressionof crestin (FIG. 2a , left and middle). The chemoinformaticDiscoverygate algorithm⁶ revealed similarity between NSC210627 andbrequinar (FIG. 5), an inhibitor of dihydroorotate dehydrogenase(DHODH)⁷. NSC210627 inhibited DHODH activity in vitro (FIG. 11).Leflunomide, a structurally distinct DHODH inhibitor⁸, phenocopiedNSC210627 (FIG. 2a , right) and was used for further studies given itsavailability.

We examined neural crest derivatives affected by leflunomide. Treatedzebrafish embryos were devoid of pigmented melanocytes at 36-48 hpf(FIG. 2a-2b ) and iridophores (data not shown) at 72 hpf. DHODHinhibition led to a loss of ventral melanocytes in stage 38 Xenopusembryos (data not shown) [Leflunomide treatment (2.5 uM) from 5-72 hpfshowed a reduction of reflective iridophores on the surface of the eye.Also microphthalmia was induced by leflunomide. Melanocytes were presentin this animal due to the lower dose of leflunomide used (data notshown). Also seen was a reduction of ventral melanocytes in Xenopusembryos treated with the DHODH inhibitor NSC210627) data not shown)].Leflunomide led to a nearly complete loss of mitf-GFP+ cells at 24 hpf(FIG. 2a-2b ), reduction of myelin basic protein (mbp)-mCherry glialcells, and jaw cartilage disruption at 72 hpf (FIG. 2a-2b and data notshown). Leflunomide reduced expression of sox10 and dct while leavingother lineages such as blood and notochord less affected (FIG. 12).Microarray analysis of leflunomide treated embryos showed downregulationof 49% of the genes upregulated in the 123-gene melanoma signature, andover half of those are neural crest related (data not shown).

The loss of multiple neural crest derivatives suggested that leflunomideacts on neural crest stem cells. We tested leflunomide, and itsderivative A771726, on neural crest stem cells (NCSCs) isolated from thefetal (E14.5) rat gut^(9,10). Both reduced the number of self-renewingNCSCs from primary stem cell colonies to 27+/−5.35% and 35+/−6.16% ofcontrols (p<0.0003 and p<0.00007, t-test, FIG. 6a ). Colony size wasreduced compared to controls (by 18% and 24%, respectively, p<0.02,t-test) but there was no effect on differentiation or survival ofspecific progeny (FIG. 6b , FIG. 6c ). These results demonstrate thatDHODH inhibitors negatively regulate NCSC self-renewal and affect NCSCsfrom multiple species.

DHODH is the fourth step in the synthesis of pyrimidine nucleotides(NTPs)¹¹. We noted striking morphological similarity between leflunomidetreated embryos and the spt5/spt6 mutants¹², suggesting that leflunomideacted to suppress transcriptional elongation. We found a lack of crestinexpression and pigmented melanocytes (similar to leflunomide) in thespt5^(sk8) null mutant (FIG. 13a ). The expression profiles of 24 hpfspt5^(sk8) mutants and leflunomide treated embryos¹³ were nearlyidentical; of 223 genes downregulated after leflunomide treatment, 183of these were similarly downregulated in spt5^(sk8) (data not shown andFIG. 13b ) including neural crest genes (crestin, sox10, mitf) andmembers of the notch pathway (her2, dlb). We examined the interaction ofDHODH with spt5 using low-doses of leflunomide (3-5 uM) incubated withthe hypomorphic spt5^(m806) mutant (which has only mild melanocytedefects)¹⁴ and then analyzing the number of pigmented melanocytes.spt5^(m806) embryos showed enhanced sensitivity to leflunomide (FIG. 3aand FIG. 7); at 3 uM, 99% of mutant embryos had few or no melanocytescompared to 0% of wild type embryos (Kruskal-Wallis, p=0.000018). Thesedata confirm that DHODH inhibition impacts transcriptional elongation,consistent with previous data demonstrating that reduction in nucleotidepools in vitro leads to defects in elongation¹⁵.

We assessed whether leflunomide specifically caused defects in thetranscriptional elongation of genes required for neural crestdevelopment using qRT-PCR (FIG. 13c and data not shown). Leflunomidecaused no change or an increase of 5′ transcript abundance, but asignificant downregulation of 3′ transcripts of mitf (5′:3.75+/−1.19 vs.3′: 0.39+/−0.07 fold, p<0.05), and dlb (5′:1.13+/−0.14 vs.3′:0.74+/−0.07 fold, p<0.05), but not in control genes such asbeta-actin (5′:1.03+/−0.07 vs. 3′:0.99+/−0.06 fold, p=NS, t-test). Inthe presence of leflunomide, transcription is initiated normally, butthese transcripts accumulate and do not undergo productive elongation.

To confirm that this mechanism is conserved in human melanoma, weperformed chromatin immunoprecipitation using an antibody to RNApolymerase II (Pol II), followed by sequencing (ChIP-seq).Transcriptional elongation was measured using the traveling ratio, TR¹⁶,where the ratio of Pol II density in the promoter-proximal region iscompared to the gene body. In both A375 and MAMLE-3M cells, leflunomidecaused a significant inhibition of transcriptional elongation (measuredas an increase in the TR), particularly for genes with an initially lowTR<7.5. For example, in A375, the TR increased by >1.3 fold in 21.3% ofloci; in MAMLE-3M, this was 36.3% of loci (data not shown). Examinationof pol II occupancy using metagene analysis at a variety of fold-changecutoffs (FIG. 3b [A375], FIG. 14 [MAMLE-3M], and data not shown)revealed no defect in transcription initiation, but a decrease inelongation pronounced at the 3′ end of genes such as Npm1 and Ccnd1(FIG. 3c ). Ingenuity Pathway Analysis on the loci affected in both celllines revealed a strong enrichment for c-Myc targets and pathwaymembers¹⁷ (data not shown). c-Myc, in addition to its requirement inneural crest development,¹⁸ was recently shown to be a potent regulatorof transcriptional pause release in ES cells¹⁶. Our data suggests theregulation of c-Myc target genes at the transcriptional elongation levelis an operative mechanism in neural crest-derived melanoma as well.Taken together, the genetic and biochemical data demonstrate thatleflunomide acts to modulate transcriptional elongation in both neuralcrest development and human melanoma.

Given the effect of DHODH inhibition on neural crest development, wetested its effects on melanoma growth. A771726 caused a dose-dependentdecrease in proliferation of human melanoma cell lines (A375, Hs.294t,RPMI7951; FIG. 4A). Similarly, an shRNA against DHODH led to a 57.7%decrease in proliferation of A375 cells, as well as a decrease inelongation as measured by ChIP-PCR (FIG. 15). Microarray analysis of theA375 cell line treated with leflunomide revealed downregulation of genesrequired for neural crest development (i.e. snai2) and members of thenotch pathway (e.g. hes6, jag1), consistent with the effects in embryos(data not shown).

NTP production is regulated at the level of carbamoyl-phosphatesynthetase (CAD)¹⁹, the enzyme upstream of DHODH. CAD is phosphorylatedby MAP kinase²⁰, which would be activated in melanoma due to theBRAF^(V600E) mutation. We reasoned that combined blockade ofBRAF^(V600E) and DHODH would cooperate to suppress melanoma growth. Wemeasured melanoma proliferation utilizing the BRAF^(V600E) inhibitorPLX4720²¹ together with A771726 (FIG. 4b , FIG. 4c and FIG. 8a , FIG. 8b), and found that the combination led to a cooperative suppression ofmelanoma growth. PLX4720 had no effect in non-melanoma cell lines(BRAF^(WT), FIG. 8c ). A771726 demonstrated mild antiproliferativeactivity but was less potent in these cells (FIG. 8d ).

We examined the in vivo effects of leflunomide and PLX4720 usingxenografts of A375 melanoma cells transplanted into nude mice (FIG. 4cand FIG. 16). At 12 days post-treatment, DMSO tumors grew7.4+/−1.3-fold, compared to 5.7+/−0.16-fold with PLX4720 and4.7+/−0.12-fold with leflunomide. The combination of PLX4720 andleflunomide led to an enhanced abrogation of tumor growth, with only2.2+/−0.9-fold growth, and in 40% of animals led to nearly completetumor regression (p<0.001, PLX/LEF vs. PLX or LEF alone, ANOVA followedby Tukey's LSD). At clinically meaningful doses, we find that aninhibitor of embryonic neural crest development, leflunomide, blocks invivo tumor growth in combination with the oncogenic BRAF^(V600E)inhibitor PLX4720.

Our data suggest that inhibition of DHODH abrogates transcriptionalelongation of genes required for both neural crest development andmelanoma growth, including mitf and myc targets. Although DHODHinhibition would be expected to lead to ubiquitous defects, humanmutations in DHODH cause Miller's syndrome²², a craniofacial disordersimilar to syndromes with defects in neural crest development. Our datasupport recent findings that elongation factors are important for bothneural crest development²³ and cancer growth²⁴. Developmental regulatorsof transcriptional elongation have recently been identified inhematopoiesis²⁵, and identification of such factors in the neural crestawaits further study.

Chemical genetic approaches in zebrafish and Xenopus allow foridentification of molecules that require in vivo contexts for theexpression of relevant phenotypes²⁶. Inhibition of DHODH may provide aunique in vivo mechanism for modulating transcriptional elongation.Leflunomide is a well-tolerated arthritis drug in humans²⁷, and our datasuggest it would be most effective incombination with a BRAF^(V600E)inhibitor. This may help to overcome resistance to BRAF^(V600E)inhibitors²⁸. As an increasing number of genomic changes are identifiedin cancer, the challenge is to target these in concert withlineage-specific factors to uncover therapeutic synergy. Our approach toidentifying lineage-specific suppressors in zebrafish embryos can begeneralized to other cell types, with direct relevance to human cancer.

Methods Summary

Microarray analysis was performed on 4 groups of 72 hpf embryos: 1) WT,2) mitf-BRAF^(V600E), 3) p53^(−/−) or 4) mitf-BRAF^(V600E);p53^(−/−)Arrays were similarly performed on adult mitf-BRAF^(V600E);p53^(−/−)melanomas and adjacent skin. The transcriptional signature of themelanomas was used in GSEA to identify genes significantly enriched inthe mitf-BRAF^(V600E);p53^(−/−) embryos. This signature (123 genes),enriched for markers of the neural crest, were concordantlyup/downregulated in both BRAF^(V600E);p53^(−/−) embryos and tumors. Insitu hybridization (ISH) for crestin (a pan NC marker) and other NCgenes, was examined in embryos (24-72 hpf) and adult tumors. Chemicalscreening was performed to identify suppressors of the crestin⁺ lineageby treating wild-type embryos from 50% epiboly to 24 hpf, followed byrobotic ISH. Two inhibitors of DHODH abrogated crestin expression:NSC210627 and leflunomide. The latter was used for further studies dueto more widespread availability. The effect of leflunomide on zebrafishembryonic neural crest development was assessed by scoring for embryonicmelanocytes, iridophores, and glial cells. Leflunomide was furtherassessed for its ability to affect multipotent self-renewal of purifiedp75⁺α₄ ⁺ rat neural crest stem cells. The effects of leflunomide ontranscriptional elongation in the neural crest was tested using thespt5^(m806) allele, and measuring pigmentation in response to 3-5 uMleflunomide. Elongation in melanoma cells was assayed by ChIP-Seq usingan antibody to RNA Polymerase II and measuring the traveling ratio.Leflunomide was tested for anti-melanoma effects in human melanoma cellslines in the presence or absence of the BRAF^(V600E) inhibitor PLX4720.In vitro proliferation assays were performed using the CellTiterGlosystem (Promega). In vivo effects were tested by treatment ofestablished A375 xenografts by daily intraperitoneal dosing of PLX4720alone, leflunomide alone or the combination, and tumor growth ratemeasured on day 4, 7 and 12.

Generation of Zebrafish Lines

Wild-type strains used in these studies were the Tu variety. Thep53^(−/−) and transgenic mitf-BRAF^(V600E) lines were established aspreviously described^(29,30). Double homozygous animals were genotypedusing PCR primers for WT BRAF (Forward: TGCTCTTGACCTCAGACTGG (SEQ IDNO:6), Reverse: CCTCAATAAACACCCTACGG)(SEQ ID NO:7), BRAF V600E (Forward:GAGGCTTTGTCGAATCGGACCGGTG (SEQ ID NO:8), Reverse:TTGAACAGAGCCTGGCCCGGCT) (SEQ ID NO:9), and p53 (Forward:TGTGTCTGTCCATCTGTTTAACAGTCA (SEQ ID NO:10), Reverse:GATAGCCTAGTGCGAGCACACTCTT) (SEQ ID NO:11), The four genotypes used forthe microarray analyses were produced by incrossing of each respectiveline.

RNA Preparation

Embryos: Fertilized eggs were collected immediately after mating, andplaced at 28.5 C. Chorions were removed by pronase solution (2.5 mg/mlin E3 water), and embryos allowed to grow until either 24 hpf or 3 dpf.Clutches containing ˜50 embryos were homogenized in Trizol, RNAprecipitated with chloroform/isopropanol, DNaseI treated and the cleanedup using RNEasy columns. RNA was then utilized for Affymetrix ZebrafishGenome chips.

Adults: A single melanoma was dissected from the head, dorsum or tail ofan adult BRAF^(V600E);p53^(−/−) fish (˜6 months of age). Extraneoustissue was removed. Simultaneously, the melanized skin directly adjacentto each given tumor was excised and acted as the comparator for thatgiven fish. Tissues were prepared identically to the embryo RNApreparations.

Microarray Analysis

BRAF^(V600E);p53^(−/−) signature: To identify genes enriched between theBRAF^(V600E);p53^(−/−) embryos and tumors, we initially identified genesthat were at least 5-fold up or downregulated in the melanomas comparedto the adjacent melanized skin. Significant genes were identified withWelch's T-test followed by Benjamini-Hochberg F D R at a q value <0.05.The top 200 up or down regulated genes were then used as the genesignature for GSEA (version 2), queried against the entire gene set(15,617 genes) for WT, p53, BRAF or BRAF^(V600E);p53^(−/−) embryos.Genes that were significantly enriched in the BRAF^(V600E);p53^(−/−)embryos compared to the other 3 strains were identified using weightingenrichment, the Signal2Noise ranking metric, and permutation=1000.

Leflunomide and spt5 signature: Tu embryos were treated with 6.5 uMleflunomide from 50% epiboly to 24 hpf, and then chorions removed andRNA extracted as above. Gene that were 2-fold up or downregulated byleflunomide were identified and then significance assessed by Welch'sT-test followed by Benjamini-Hochberg FDR. This yielded 223downregulated and 60 upregulated genes, which were then used in GSEAagainst a previously published spt5 (sk8) null mutant signature. Genesenriched between the leflunomide treatment and spt5 null mutants wereidentified using the same GSEA algorithm as above.

A375 signature: A375 cells were treated with 25 uM leflunomide for 3days, and RNA then isolated using Trizol as above. Expression analysiswas performed using Illumina WG6 v3 arrays, and the data was analyzedusing Illumina Genome Studio. Genes significantly affected byleflunomide are those with an Illumina difference score of +/−30 orgreater, which corresponds to a p value<0.001.

Chemical Screening Methodology

The screen was performed essentially as previously described³¹. Inbrief, WT embryos were collected, staged and then plated at a density of10 embryos per well in a well-well tissue culture plate in 200 ul of E3water. At 50% epiboly, 100 ul of a 3× chemical stock was added to eachwell using a Hamilton liquid handling robot, yielding a final 1×concentration. For the majority of the chemicals in the library, thisyielded a concentration of −33 uM. The libraries used were the BIOMOL480, Sigma LOPAC 1280, and selected additional compounds obtainedthrough the Children's Hospital Boston Chemical Screening facility.Compounds known to affect pigmentation in Xenopus were obtained from ascreen previously described³²′³³. After treatment, embryos were rinsedin E3 water, pronase treated to remove chorions, washed 4× in E3 and 1×in PBT, and then fixed in 4% PFA for 24 hours. Fixed embryos werechanged to 100% methanol 24 h later, and kept until the time of ISH.

Embryos underwent automated ISH using a Biolane HTI robot with a crestinriboprobe as previously described³¹. All wells were manually scored byeye, assigning a score within a range of 1 (no crestin staining) to 3(no change in crestin) to 5 (marked increase in crestin). Note was alsotaken of chemicals that affected migratory patterns in both A-P and D-Vdirections, and overall morphology. Approximately 7% of chemicals weregenerally toxic, 2.2% showed a decrease in crestin, and 1% showed anincrease in crestin. All hits were validated in larger, independentclutches of 100-200 embryos using chemicals obtained from independentsuppliers.

In Vitro DHODH Assay

The DHODH activity assay monitored the reduction of2,6-dichloroindophenol (DCIP) and was conducted in 50 μl of 100 mM HEPES(pH 8.0) containing 150 mM NaCl, 5% glycerol, 0.05% Triton X-100, 175 μML-DHO, 18 μM decylubiquinone (CoQD), and 95 μM DCIP, arrayed in a384-well format. The concentrations of enzymes used were as follows: P.falciparum 12.5 nM, human 7 nM. Following a 20 minute incubation at roomtemperature the absorbance was measured at 600 nm (Envision,PerkinElmer). A sigmoidal dose-response curve was generated by plotting% inhibition as a function of the log of compound concentration (range:1.5 nM to 30 μM), and an IC50 value representing the concentration atwhich inhibition was half-maximal was determined.

Zebrafish Neural Crest Assay

For melanocyte assays, embryos were treated with leflunomide 5-6.5 uMfrom 50% epiboly until 48 hpf, at which point melanocytes along thedorsal axis and ventral pathways were assessed using visual scoring. Themitf-GFP transgenic was scored at 24 hpf, as this is the point ofmaximal GFP intensity. For the iridophore, mbp-mCherry and jaw cartilageexperiments, the embryos were treated with 2.5 uM leflunomide to allowthem to live beyond 48 hpf.

Rat Neural Crest Stem Cell Assays

Adherent Culture of NCSC

Enteric NCSCs from the fetal (E14.5) rat gut were isolated using flowcytometry to select the 1%-2% of cells expressing the highest levels p75and α4 integrin as previously described⁹. Cells were plated at clonaldensity in two wells/treatment and cultured in self-renewal medium for 8days in LEF, A771726 or the vehicle (DMSO) followed by differentiationmedium+/−LEF, A771726 or DMSO for 6 days. Cultures were fixed in 4% PFAthen stained with antibodies against peripherin, GFAP, and SMA toidentify neurons (N), glia (G) and myofibroblasts (M) respectively(U=unstained cells). A total of 3 to 4 independent experiments wereperformed, and differences measured by ANOVA followed by Bonferonniposthoc comparison. Self-renewal medium contains: DMEM-low glucose, 15%Chick Embryo Extract (CEE), Penn/Strep (P/S), 1% N2 supplement, 2% B27supplement, 110 nM Retinoic Acid, 50 μM 2-mercaptoethanol 20 ng/ml bFGF,and 20 ng/ml IGF-1. Differentiation medium is the same except itcontains 1% CEE, 10 ng/ml bFGF and no IGF-1. Differences between groupswere calculated using ANOVA followed by posthoc Sheffe.

Non-Adherent Culture of NCSC

Dissociated fetal rat gut cells were plated at clonal density innon-adherent conditions in self-renewal medium with LEF, a77 or DMSO for10 days¹⁰. Three to five primary neurospheres per treatment wereindividually re-plated in adherent conditions with LEF, a77 or DMSO andafter three days were dissociated into a single cell suspension usingtrypsin and collagenase IV. Dissociated cells from each neurosphere werere-plated in non-adherent conditions without LEF or a77 and after 10days, the number of secondary neurospheres were counted and compared. Toassess the number of multipotent primary and secondary neurospheres, asubset of neurospheres were plated adherently for 8 days in self-renewalmedium followed by 6 days in differentiation medium. Cultures were fixedin 4% PFA and stained with antibodies against peripherin, GFAP, and SMAto identify neurons (N), glia (G) and myofibroblasts (M) respectively. Atotal of 5 independent experiments were analyzed, and the number ofmultipotent secondary neurospheres on a given day are normalized to theDMSO control wells, and differences calculated by ANOVA.

spt5 Hypomorphic Experiments

The spt5^(m806) allele was used to assess whether leflunomide interactedwith the elongation machinery. This allele has only a mild pigmentdefect on its own. Heterozygous spt5^(m806) adults were incrossed, andembryos then collected and treated with leflunomide 3, 4 or 5 uM, asabove. Embryos were scored at 48 hpf for pigmentation, scored as“unpigmented”, “light”, or “fully pigmented” as compared to untreatedwells. Individual embryos were then genotyped for the spt5^(m806)mutation as previously described¹⁴. Kruskall-Wallis analysis was used todetect a difference between the genotyped groups.

5′-3′ Transcriptional Elongation Assays

Embryos were treated with DMSO or leflunomide 6.5 uM from 50% epibolyuntil 24 hpf, and then RNA prepared as above. Primers were designed tothe noted genes, situated at the very 5′ end or 3′ end of the gene,situated in the UTR if annotated. qRT-PCR was then performed using SYBRGreen, and each primer pair was normalized to an average of the Ct valuefor the 5′ beta-actin and 3′ beta-actin primers. Fold-change wascalculated using the delta-delta Ct method, and all values are expressedrelative to the DMSO control, after log 2 transformation. Differencesbetween groups were assessed using t-tests. Primer sequences availableupon request.

RNA Pol II ChIP-Seq Analysis Following Leflunomide Treatment

Chromatin Immunoprecipitation

RNA Pol II ChIP-seq analysis was done in A375 and MAMLE-3M cells withand without Leflunomide treatment as described in¹⁶. In brief, A375 andMAMLE-3M cells were grown as described above and treated with 25 uM(A375) or 50 uM (MAMLE-3M) leflunomide (dissolved in DMSO) or DMSO alonefor 72 hours. Cells were crosslinked for 10 minutes with the addition ofone-tenth of the volume of 11% formaldehyde solution (11% formaldehyde,50 mM Hepes pH7.3, 100 mM NaCl, 1 mM EDTA pH8.0, 0.5 mM EGTA pH8.0) tothe growth media followed by two washes with PBS. Cells were scraped andfrozen in liquid nitrogen. 100 ul of Dynal magnetic beads (Sigma) wereblocked with 0.5% BSA (w/v) in PBS. Magnetic beads were bound with 10 ugof total Pol II antibody (Rpb1 N-terminus, Santa Cruz sc-899). Unboundantibody was washed away following an overnight incubation at 4° C.Crosslinked cells were lysed with lysis buffer 1 (50 mM Hepes pH7.3, 140mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, and 0.25% Triton X-100)and washed with lysis buffer 2 (10 mM Tris-HCl pH8.0, 200 mM NaCl, 1 mMEDTA pH8.0 and 0.5 mM EGTA pH8.0). Cells were resuspended and sonicatedin sonication buffer (50 mM Tris-HCl pH7.5, 140 mM NaCl, 1 mM EDTA, 1 mMEGTA, 1% Triton X-100, 0.1% Na-deoxycholate, 0.1% SDS) for 8 cycles at30 seconds each on ice (18 watts) with 60 seconds on ice between cycles.Sonicated lysates were cleared and incubated overnight at 4° C. withmagnetic beads bound with antibody to enrich for DNA fragments bound bythe indicated factor. Beads were washed three times with sonicationbuffer, one time with sonication buffer with 500 mM NaCl, one time withLiCl wash buffer (20 mM Tris pH8.0, 1 mM EDTA, 250 mM LiCl, 0.5% NP-40,0.5% Na-deoxycholate) and one time with TE. DNA was eluted in elutionbuffer. Cross-links were reversed overnight. RNA and protein weredigested using RNAse A and Proteinase K, respectively and DNA waspurified with phenol chloroform extraction and ethanol precipitation.The purified DNA from A375+Leflunomide, A375+DMSO, A375 input DNA,Mamle+Leflunomide, Mamle+DMSO and Mamle input DNA samples were then usedfor preparation of Illumina sequencing libraries.

Sample Preparation

Purified DNA was prepared for sequencing according to a modified versionof the Illumina/Solexa Genomic DNA protocol. Fragmented DNA was preparedby repairing the ends and adding a single adenine nucleotide overhang toallow for directional ligation. A 1:30 dilution (in water) of theAdaptor Oligo Mix (Illumina) was used in the ligation step. A subsequentPCR step with limited (18) amplification cycles added additional linkersequence to the fragments to prepare them for annealing to the GenomeAnalyzer flow-cell. The amplified library was size selected byseparation on a 2% agarose gel and a band between 150-300 bp(representing shear fragments between 50 and 200 nt in length and ˜100bp of primer sequence) was excised. The DNA was purified from theagarose and this DNA library was subsequently used for polony generationand sequencing.

Polony Generation and Sequencing

The DNA library (2-4 pM) was applied to the flow-cell (8 samples perflow-cell) using the Cluster Station device from Illumina. Theconcentration of library applied to the flow-cell was calibrated suchthat polonies generated in the bridge amplification step originate fromsingle strands of DNA. Multiple rounds of amplification reagents wereflowed across the cell in the bridge amplification step to generatepolonies of approximately 1,000 strands in 1 μm diameter spots. Doublestranded polonies were visually checked for density and morphology bystaining with a 1:5000 dilution of SYBR Green I (Invitrogen) andvisualizing with a microscope under fluorescent illumination. Validatedflow-cells were stored at 4° C. until sequencing.

Flow-cells were removed from storage and subjected to linearization andannealing of sequencing primer on the Cluster Station. Primed flow-cellswere loaded into the Illumina Genome Analyzer 1G. After the first basewas incorporated in the Sequencing-by-Synthesis reaction the process waspaused for a key quality control checkpoint. A small section of eachlane was imaged and the average intensity value for all four bases wascompared to minimum thresholds. Flow-cells with low first baseintensities were re-primed and if signal was not recovered the flow-cellwas aborted. Flow-cells with signal intensities meeting the minimumthresholds were resumed and sequenced for 26 or 32 cycles.

Solexa Data Analysis

Images acquired from the Illumina/Solexa sequencer were processedthrough the bundled Solexa image extraction pipeline, which identifiedpolony positions, performed base-calling and generated QC statistics.Sequences were aligned using ELAND software to NCBI Build 36 (UCSC hg18)of the human genome. Only sequences that mapped uniquely to the genomewith zero or one mismatch were used for further analysis. When multiplereads mapped to the same genomic position, a maximum of two readsmapping to the same position were used. Analysis methods were derivedfrom previously published methods³⁴⁻³⁷. Each read was extended 100 bp,towards the interior of the sequenced fragment, based on the strand ofthe alignment. The number of ChIP-Seq reads across the genome, in 25 bpbins within a 1 kb window surrounding each bin (+/−500 bp) wastabulated. The 25 bp genomic bins that contained statisticallysignificant ChIP-Seq enrichment was identified by comparison to aPoissonian background model. Assuming background reads are spreadrandomly throughout the genome, the probability of observing a givennumber of reads in a 1 kb window can be modeled as a Poisson process inwhich the expectation can be estimated as the number of mapped readsmultiplied by the number of bins (40) into which each read maps, dividedby the total number of bins available (estimated at 70%). Enriched binswithin 1 kb of one another were combined into regions.

The Poissonian background model assumes a random distribution ofbackground reads, however we have observed deviations from thisexpectation. Some of these non-random events can be detected as sites ofapparent enrichment in negative control DNA samples and can create manyfalse positives in ChIP-Seq experiments. To remove these regions whendetermining enriched regions, we compared genomic bins and regions thatmeet the statistical threshold for enrichment to a set of reads obtainedfrom Solexa sequencing of DNA from whole cell extract (WCE) in matchedcell samples. We required that enriched bins and enriched regions havefive-fold greater ChIP-Seq density in the specific IP sample, comparedwith the control sample, normalized to the total number of reads in eachdataset. This served to filter out genomic regions that are biased tohaving a greater than expected background density of ChIP-Seq reads. Forcomparison of Pol II occupancy following Leflunomide treatment with acontrol dataset, rank normalization was used to normalize the datasetsbeing compared. This normalization method is described in³⁸. Briefly, aquantile normalization method was used for analysis. For each datasetcompared, the genomic bin with the greatest ChIP-Seq density wasidentified. The average of these values was calculated and the highestsignal bin in each dataset was assigned this average value. This wasrepeated for all genomic bins from the greatest signal to the least,assigning each the average ChIP-Seq signal for all bins of that rankacross all datasets.

Traveling Ratio Calculation

Traveling ratios (TR) were calculated as described in¹⁶. Briefly, TRcompares the ratio between Pol II density in the promoter and in thegene body region. The promoter region is defined as −300 bp to +300 bprelative to the TSS and the gene body is defined as the remaining lengthof the gene plus 2500 bp relative to the gene end. We chose to use thegene body plus the 2.5 kb after the gene end in our TR calculationsbecause we have noticed both here and in Rahl et al, that changes inelongating Pol II density are most pronounced just after the end of thegene, presumably in the transcription termination region.

Meta Gene Analysis

In order to quantify the effects of Leflunomide treatment on thepromoter and gene body regions, we first created density vectors for allgenes. The densities in the promoter, gene body, and gene end regionwere placed into 360 bins to create a density vector. 80 fixed widthbins for the −2000 to 0 promoter region, 200 variable bins equallydividing the length of the gene, and 80 fixed bins for the 0 to +2 kbafter transcript end region. For each gene, the promoter density vectorwas defined as the density in the 12 bins upstream of the TSS plus thedensity in the 6 variable length bins downstream. The gene body densityvector was defined as the density in the last 160 variable length binscomprising the gene plus the 80 fixed width bins after the gene end.

To quantify the effects of Leflunomide treatment, the matrix of densityvectors for a given gene set in either DMSO or Leflunomide treated cellswas compared using a Welch's t-test. This was performed for genes withTR changes at various fold-change cut offs in both A375 and MAMLE cells,with comparison of pol II in the promoter or gene body of loci at eachfold change cutoff.

The method used here is a variation of the analysis used in Rahl et al,and is more sensitive due to the fact that gene densities are treated asvectors rather than mean values.

Prediction of c-Myc Target Gene Enrichment in ChIP-Seq Data

The genes most inhibited in elongation were entered into IngenuityPathway Analysis (IPA) software as per manufacturer's instructions.Pathway analysis, which included the myc module, was performed as partof standard IPA analysis. For identification of c-myc target genescontained within Supplemental Table 4, we utilized the data found in¹⁷.

ChIP-PCR

ChIP for RNA polymerase II was carried out as above using A375-scrambleand A375-shRNA#877 cells. For the shRNA against DHODH, we utilized TRNCclone TRCN0000025839 (sequence:CCGGCGATGGGCTGATTGTTACGAACTCGAGTTCGTAACAATCAGCCCATCGTTTTT (SE ID NO:12).A375 cells were infected with 200 ul of viral supernatant in 2 ml ofmedia with protamine, and cells selected with puromycin. Cells weregrown to 60% confluency and crosslinked and harvested with formaldehyde.ChIP DNA was obtained for both IgG control as well as pol II (Santa Cruzsc-899). PCR primers were designed based on the peaks seen in ChIP-Seqdata from the DMSO and leflunomide assays. qPCR using SYBR GreenER(Invitrogen) was performed for 50 cycles with an extension/read time of60 seconds at 60 C. The fold enrichment for the sh877 compared toscramble cells was calculated as2^[(C_(t)Pol2−C_(t)IgG)_(scramble)−(C_(t)Pol2−C_(t)IgG)_(sh877)].

In Vitro Melanoma Proliferation Assays

Human melanoma cell lines (A375, RPMI7951, Hs.294T, MAMLE-3M) wereobtained from the American Type Culture Collection. The 8988 cell linewas provided by Alec Kimmelman at Dana Farber, and the PANC1 andMDA-231-MB lines were provided by Rania Hartouche at Harvard. Cells werecultured in DMEM/10% FCS at 37 C, 5% CO₂. For proliferation assays,96-well clear bottom plates were used to plate cells at a density of5,000 cells/well (RPMI7951 and A375) or 10,000 cells/well (MAMLE-3M-3Mand Hs.294T) in 100 ul containing indicated concentrations of A771726 orPLX4720 or DMSO. All conditions were tested in triplicate. Media waschanged at day 2, and proliferation rate was measured at day 4 using theCellTiterGlo assay kit (Promega). All values are expressed are the % ofluciferase relative to the DMSO control well. Differences between groupswere statistically analyzed using a generalized linear ANOVA modeltaking into account factors of PLX4720, A771726 and dose. IC50's werecalculated using a log-logistic model.

For proliferation assays in the A375-scramble and A375-sh877 cells,50,000 cells were plated on day 0 and grown for the indicated time inpuromycin containing media. Cells were harvested (in triplicate) andcounted with a hemocytometer on days 4, 6 and 8.

In Vivo Xenograft Studies

The A375 cell line was chosen for xenografts based on prior experiencewith PLX4720 in this line. Cells were grown to 60-70% confluence,trypsinized and 3.5×10⁵ cells were transplanted into the right flank ofnude mice in a volume of 200 ul. Tumors were allowed to develop for 2weeks (day 0) at which time daily intraperitoneal dosing of either DMSO,PLX4720 2.5 mg/kg, Leflunomide 7.5 mg/kg or both PLX4720/Leflunomide wasbegun. Tumors were measured with digital calipers on day 4, 7 and 12 andgrowth rate was compared to the tumor size on day 0. Growth rates werecompared between DMSO, PLX, LEF or PLX4720 using ANOVA with repeatedmeasures to identify significant interactions. For the day 12 data,differences between the groups were compared using ANOVA followed byposthoc Tukey's least-squared difference.

Human Tissue Array

Human melanoma array ME 1001 (US Biomax) was stained using antibodies toednrb (Abcam ab1921), edn3 (PTG Inc. 10674-1-AP), mitf (Abcam 3201) ordct (Sigma HPA010743). Melanomas were scored by an independentpathologist (SG) for reactivity to given antibody. In some cases, tumorsstained for multiple antibodies in different areas of the same tumor,and these were scored as positive for each individual antigen.

All the references cited below and cited throughout the specification,are herein incorporated by reference in their entirety.

EXAMPLE REFERENCES

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Biochemistry 40,    2194-2200 (2001).-   8. Kaplan, M. J. Leflunomide Aventis Pharma. Curr. Opin. Investig    Drugs 2, 222-230 (2001).-   9. Bixby, S., Kruger, G. M., Mosher, J. T., Joseph, N. M. &    Morrison, S. J. Cell-intrinsic differences between stem cells from    different regions of the peripheral nervous system regulate the    generation of neural diversity. Neuron 35, 643-656 (2002).-   10. Molofsky, A. V. et al. Bmi-1 dependence distinguishes neural    stem cell self-renewal from progenitor proliferation. Nature 425,    962-967 (2003).-   11. Loffler, M., Jockel, J., Schuster, G. & Becker, C.    Dihydroorotat-ubiquinone oxidoreductase links mitochondria in the    biosynthesis of pyrimidine nucleotides. Mol. Cell. Biochem. 174,    125-129 (1997).-   12. Keegan, B. R. et al. The elongation factors Pandora/Spt6 and    Foggy/Spt5 promote transcription in the zebrafish embryo.    Development 129, 1623-1632 (2002).-   13. Krishnan, K., Salomonis, N. & Guo, S. Identification of Spt5    target genes in zebrafish development reveals its dual activity in    vivo. PLoS One 3, e3621 (2008).-   14. Guo, S. et al. A regulator of transcriptional elongation    controls vertebrate neuronal development. Nature 408, 366-369    (2000).-   15. Wada, T. et al. DSIF, a novel transcription elongation factor    that regulates RNA polymerase II processivity, is composed of human    Spt4 and Spt5 homologs. Genes Dev. 12, 343-356 (1998).-   16. Rahl, P. B. et al. c-Myc regulates transcriptional pause    release. Cell 141, 432-445 (2010).-   17. Chen, Y. et al. Integration of genome and chromatin structure    with gene expression profiles to predict c-MYC recognition site    binding and function. PLoS Comput. Biol. 3, e63 (2007).-   18. Hong, S. K., Tsang, M. & Dawid, I. B. The mych gene is required    for neural crest survival during zebrafish development. PLoS One 3,    e2029 (2008).-   19. Aoki, T., Morris, H. P. & Weber, G. Regulatory properties and    behavior of activity of carbamoyl phosphate synthetase II    (glutamine-hydrolyzing) in normal and proliferating tissues. J.    Biol. Chem. 257, 432-438 (1982).-   20. Graves, L. M. et al. Regulation of carbamoyl phosphate    synthetase by MAP kinase. Nature 403, 328-332 (2000).-   21. Tsai, J. et al. Discovery of a selective inhibitor of oncogenic    B-Raf kinase with potent antimelanoma activity. Proc. Natl. Acad.    Sci. U.S.A. 105, 3041-3046 (2008).-   22. Ng, S. B. et al. Exome sequencing identifies the cause of a    mendelian disorder. Nat. Genet. 42, 30-35 (2010).-   23. Nguyen, C. T., Langenbacher, A., Hsieh, M. & Chen, J. N. The    PAF1 complex component Leo1 is essential for cardiac and neural    crest development in zebrafish. Dev. Biol. 341, 167-175 (2010).-   24. Mueller, D. et al. Misguided transcriptional elongation causes    mixed lineage leukemia. PLoS Biol. 7, e1000249 (2009).-   25. Bai, X. et al. TIF1 gamma controls erythroid cell fate by    regulating transcription elongation. Cell 142, 133-143 (2010).-   26. Wheeler, G. N. & Brandli, A. W. Simple vertebrate models for    chemical genetics and drug discovery screens: lessons from zebrafish    and Xenopus. Dev. Dyn. 238, 1287-1308 (2009).-   27. Schiff, M. H., Strand, V., Oed, C. & Loew-Friedrich, I.    Leflunomide: efficacy and safety in clinical trials for the    treatment of rheumatoid arthritis. Drugs Today (Barc) 36, 383-394    (2000).-   28. Flaherty, K. T. et al. Inhibition of mutated, activated BRAF in    metastatic melanoma. N. Engl. J. Med. 363, 809-819 (2010).-   29. Berghmans, S. et al. Tp53 Mutant Zebrafish Develop Malignant    Peripheral Nerve Sheath Tumors. Proc. Natl. Acad. Sci. U.S.A. 102,    407-412 (2005).-   30. Patton, E. E. et al. BRAF mutations are sufficient to promote    nevi formation and cooperate with p53 in the genesis of melanoma.    Curr Biol 15, 249-54 (2005).-   31. Kaufman, C. K., White, R. M. & Zon, L. Chemical genetic    screening in the zebrafish embryo. Nat. Protoc. 4, 1422-1432 (2009).-   32. Tomlinson, M. L., Rejzek, M., Fidock, M., Field, R. A. &    Wheeler, G. N. Chemical genomics identifies compounds affecting    Xenopus laevis pigment cell development. Mol. Biosyst 5, 376-384    (2009).-   33. Tomlinson, M. L. et al. A chemical genomic approach identifies    matrix metalloproteinases as playing an essential and specific role    in Xenopus melanophore migration. Chem. Biol. 16, 93-104 (2009).-   34. Mikkelsen, T. S. et al. Genome-wide maps of chromatin state in    pluripotent and lineage-committed cells. Nature 448, 553-560 (2007).-   35. Johnson, D. S., Mortazavi, A., Myers, R. M. & Wold, B.    Genome-wide mapping of in vivo protein-DNA interactions. Science    316, 1497-1502 (2007).-   36. Marson, A. et al. Connecting microRNA genes to the core    transcriptional regulatory circuitry of embryonic stem cells. Cell    134, 521-533 (2008).-   37. Guenther, M. 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Example II

Example guidance for a Phase II trial to test the efficacy of acombination therapy of the combination therapy of BRAF inhibitor PLX4032and leflunomide.

We have found Leflunomide to block neural crest cell fate by inhibitingtranscriptional elongation, wherein a reduced number of neural crestprogenitors leading to a decrease in the number of differentiatedmelanocyte cells unexpectedly indicated that the compound is capable ofinhibiting melanoma. The targets of these events include c-myc and mitf(FIG. 9, FIG. 13) directed genes. Prior to this finding, it was notobvious and was unexpected that Leflunomide could be utilized intherapies to treat melanoma given its previous use as an anti-arthriticagent predicated on its ability to target lymphocytes (See e.g., Fox etal., (1999) Mechanism of Action for Leflunomide in Rhuematoid Arthritis,Clinical immunology 93(3): 198-208). Non-lymphocytes were previouslybelieved to be unaffected because of their ability to meet theirribonucleotide needs utilizing the salvage pyrimidine pathway. Melanomais a non-lymphocyte derived cancer characterized by uncontrolled growthof melanocytes. Thus, the combination of the Leflunomide, which attacksmelanoma cell fate, coupled to BRAF-inhibitor that targets cellproliferation is a novel and non-obvious combination aimed to treatmelanoma. Furthermore, the fact that only subclinical dosing arerequired points to unexpected synergy, which renders this invention botha more effective and safer therapy than current approaches.

To undertake a phase II trial testing the efficacy of the neural creststem cell inhibitor leflunomide will be used in combination with theBRAF inhibitor PLX4032. Such a clinical trial will be the first totarget BRAF (e.g. cell proliferation) and to target melanoma cell fateusing leflunomide. We are the first to show that Leflunomide blocksneural crest cell fate by inhibiting transcriptional elongation (e.g.inhibiting c-myc and mitf (FIG. 9, 13) directed genes.)

Combination therapies that include BRAF inhibitors will be particularlyimportant for treating melanoma, especially within the context ofdeveloping long term responses. PLX40321 (Tsai, J. et al. Discovery of aselective inhibitor of oncogenic B-Raf kinase with potent antimelanomaactivity. Proc. Natl. Acad. Sci. U.S.A. 105, 3041-3046 (2008)), andGSK211843616 (Kefford, R. A. et al. Phase I/II study of GSK2118436, aselective inhibitor of oncogenic mutant BRAF kinase, in patients withmetastatic melanoma and other solid tumors. J. Clin. Oncol. 27, Abstract8503 (2010)), the first generation of highly selective BRAF inhibitors,have completed phase II and (in the case of PLX4032) phase III testing;both have demonstrated unprecedented short-term efficacy in patientswith metastatic melanoma that harbors a BRAF mutation. Approximately 60%of patients achieve objective responses early in the course of therapy,and approximately 90% of all patients realize some degree of tumorregression. However, most responses are not long-lasting and the overallmedian progression-free survival (PFS) is 6 to 7 months for all patientstreated. Very few patients (3-5%) achieve complete responses, but whenthey do occur they are more long lasting. Thus, one metrics for judgingimprovement in outcome for the combination regimen with BRAF inhibitorsis to improve the duration of response (PFS) or increase the percentageof patients with complete responses, by a statistically significantamount. It is expected that the combination of BRAF inhibitor PLX4032and leflunomide will significantly improve PFS, or significantlyincrease the percent of patients with complete responses.

For controls, tumor biopsy samples before, during and at progressionusing single-agents PLX and GSK2118436 will be taken and analyzed; andcompared to the combination therapy of the phase II trial that combinesleflunomide and PLX.

Leflunomide is given to patients with arthritis, at doses that producesteady state concentrations exceeding those used in our preclinicalstudies. Thus, we will start with the FDA-approved dose of leflunomide(Kaplan, M. J. Leflunomide Aventis Pharma. Curr. Opin. Investig Drugs 2,222-230 (2001)). We expect that doses much smaller than those requiredfor therapeutic efficacy in arthritis patients will be needed fortherapeutic efficacy in melanoma treatment. We will also start with thestandard dose of PLX in our proposed trial. Given the efficacy of thecombination in preclinical studies, we expect that doses smaller thanthose required for therapeutic efficacy in arthritis patients will beneeded for therapeutic efficacy in melanoma treatment.

An example dose and administration regime follows: Within 6-12 hours ofa single dose, leflunomide is metabolized into a single activemetabolite that has a long half-life (˜2 weeks). Therefore, whenleflunomide is given in 3 daily loading doses (100 mg daily for 3 days),followed by lower daily doses (20 mg per day), it produces steady-stateconcentrations of approximately 60 mcg/mL (Rozman, B. Clinicalpharmacokinetics of leflunomide. Clin. Pharmacokinet 41, 421-430(2002)). Neither the parent compound nor the active metabolite ismetabolized by CYP450 isoenzymes, so there is very little possibility ofa drug-drug interaction with PLX. In placebo-controlled clinical trialswith use of leflunomide in arthritis patients, the onlytreatment-related adverse event that was more prevalent for leflunomidethan with placebo was elevation of liver transaminase. While this isalso seen with PLX, it is rarely dose limiting. We thus have a lowconcern for leflunomide contributing to the toxicity of PLX. As isstandard practice, we will monitor liver transaminases in our patientsusing means well known to those of skill in the art.

The primary endpoint of the proposed phase II trial that combinesleflunomide with PLX will be PFS; we expect improvement in median PFSfrom 7 months (observed in phase I, II and III trials with single-agentPLX) to 10 months or more. Assuming 90% power and a type I error of 5%,this would require 43 patients. Our secondary clinical endpoint iscomplete response. We will use 90% power to detect an increase incomplete rate to 12% or higher. A critical secondary endpoint will bethe determination of pharmacodynamic effects of leflunomide in patients'tumors: to accomplish this, patients will start leflunomide as asingle-agent for two weeks, before initiating PLX. Of the 43 patients tobe accrued, we will require that 20 patients have tumors (cutaneous,subcutaneous or palpable lymph node metastases) that can be biopsiedbefore and during treatment with use of a core needle or 3-4 mm punchbiopsy. The remaining 23 patients may be accrued independently of theability to perform biopsies, and once enrolled, only patients withtumors that can be biopsied will be accrued for the remainder of thetrial. Biopsies will be performed before the initiation of treatment andbetween days 10-14 of single-agent leflunomide treatment.

Example III Additional Compounds for the Treatment of Melanoma

We assessed the ability of multiple DHODH inhibitors for their abilityto treat melanoma both in vitro and in vivo: C₁₉H₁₄F₂N₂O₃, compound 1;C₂₀H₁₆F₂N₂O₃, compound 2; C₁₈H₁₃F₆NO₄, compound 3; C₁₉H₁₆F₃NO₄, compound4; C₁₉H₁₀F₅NO₄S, compound 5; C₁₉H₁₄FNO₄S, compound 6; C₂₀H₁₅F₄NO₄,compound 7; C₁₉H₁₇NO₄, compound 8; C₂₀H₁₇F₂NO₄, compound 9; andC₂₀H₁₈FNO₄, compound 10. FIG. 21 shows the chemical structures of DHODHinhibitors compounds 1-4.

Compounds 1 and 2 were synthesized as described in U.S. PatentPublication 2010/007898 and compounds 3 and 4 were synthesized asdescribed in U.S. Pat. No. 7,074,831, each of which are hereinincorporated by reference in their entireties. FIG. 22 show the chemicalstructures of DHODH inhibitors for compounds 5 to 10. Synthesis ofcompound 5 and compound 6 is described in U.S. Patent Publication2004/0192758, which is herein incorporated by reference in its entirety.Compounds 7-10 are described in U.S. Patent Publication 2006/0199859,and in U.S. Pat. No. 7,176,241, which are herein incorporated byreference in their entirety.

We used in situ hybridization (ISH) staining in Zebrafish embryos tomonitor the suppression of neural crest progenitors in the presence ofthe various compounds as a means to assess the efficacy of the compoundsin treatment of melanoma. FIG. 24 shows the chemical structures ofleflunomide and its active metabolite A77126, and a structurallydifferent DHODH inhibitor compound 1 (inhibitor DH#1). FIG. 24b showsthe effect of compound 1, leflunomide, and A77126, on Zebrafish embryoson neural crest development, in the presence of DMSO, Decylubiquinone,Aphiadicolin, or Progesterone. ISH staining indicates that each of theseinhibitors (leflunomide, A771726, or DH #1) leads to reduction inexpression of the neural crest, depicted by an in situ hybridization forthe neural crest marker crestin. However, chemicals includingdecylubiquinone, aphidicolin, and progesterone suppress these drugs andlead to a rescue of the neural crest population.

The efficacy of compound 1, compound 7, leflunomide, compound 5 andcompound 6, was also determined by in situ hybridization (ISH) stainingin Zebrafish embryos by monitoring neural crest development in thepresence of the compounds (See FIGS. 25a to 25b ; FIGS. 21-23 forcompound structure and formula). Different concentrations of the DHODHinhibitors and in situ for crestin revealed compound 1 and compound 7(FIG. 25a (#1 and #7) impair crestin expression at lower doses comparedto leflunomide and compounds 5 and 6, FIG. 25 b #5 and #6, respectively.The compounds showed varied strength in their ability to inhibit neuralcrest development, e.g. the effect of compound 1>compound 7>leflunomide,which is approximately the same as compounds 5 and 6.

The efficacy of compound 4, compound 9 and compound 2 is shown in FIG.26 (#4, #9 and #2 respectively; See FIGS. 21 to 23 for compoundstructure and formula). When referring also to the results obtained inFIG. 25, it is shown that DHODH inhibitors #4, #9 and #2 are lesseffective than leflunomide. Indeed at 1.75 uM these drugs do not affectcrestin expression like observed upon the same dose of leflunomide inFIG. 25b . The effect of compound 1>compound 7>leflunomide, which isapproximately the same as compounds 5 and 6, which >compound 4, which isabout the same as compound 9, which >compound 10>compound 2.

We also tested the effect of all of the DHODH inhibitors compounds 1-10(DHODH analogs) (See FIGS. 21-23) on A375 cells after 3 days oftreatment, See FIG. 27 for the results. Compounds 1, 4, and 7significantly decreased cell proliferation at lower doses compared toleflunomide and A771726.

FIGS. 28a to 28e show graphs depicting the effects of leflunomide,compound 1 (PH-1), and compound 7 (PH-7) (See FIG. 21 (compound 1) andFIG. 23 (compound 7)) on the indicated melanoma cells in vitro. FIG. 28a, M481 cells. FIG. 28b , A375 cells. FIG. 28c , M405 cells. FIG. 28d ,M528 cells. FIG. 28e , M715 cells. Compound 1, compound 7 and leflumideaffect cell viability of human primary melanoma cells. FIGS. 28a and 28bshow respectively that lefunomide as well as DHODH #1 (PH-01, i.e.compound 1), DHODH#7 (PH-07, i.e. compound 7) impair cell survival inprimary human melanoma cells (M481) and in the melanoma cell line(A375). FIG. 28c shows that the effect of these inhibitors is variablein different primary melanoma cells (M405, M528 and M715).

We also tested the in vivo effects of leflunimide in combination withdigitoxin on M405 tumor xenografts in mouse. FIG. 29a , shows the effecton tumor diameter (cm) vs. time. FIG. 29b , effect on tumor weight. FIG.29c , effect on frequency of melanoma cells in the blood. FIG. 29d ,Total flux (photons/second). The M405 cells were not sensitive to drugtreatment as shown in FIG. 28 and accordingly there is no effect ontumor growth (FIG. 29a ) and number of melanoma cells in bloodcirculating cells (FIG. 29b ). But there is significant difference inblood circulating cells in co-treatment with Leflunomide and Digitoxin.No change in flux was observed (FIG. 29d ).

In addition, M841 primary human melanoma xenografts in mice revealedthat leflunomide and other DHODH inhibitors are more effective when usedin combination with Plexicon (FIG. 30 and data not shown). FIG. 30 showsthe in vivo effects of the combination of DHODH inhibitor and Brafinhibitor on tumor volume, of M481 primary human melanoma xenografts inmice.

We have also determined that Leflunomide and other DHODH inhibitorssignificantly impair tumor growth in combination with BRAFi and MEKi(FIG. 31 and Data not shown). FIG. 31 shows the effect leflunomide incombination with Braf and Mek inhibitors on primary human melanoma cellsxenografts in mice. Human BRAFV600 (M491) cells were treated with acombination of drugs including BRAF inhibitors (BRAFi), MEK inhibitors(MEKi) and Leflunomide.

SEQUENCES Human wt BRAF (SEQ ID NO: 1)   1 maalsggggg gaepgqalth gdmepeagag agaaassaad paipeevwni kqmikltqeh  61 iealldkfgg ehnppsiyle ayeeytskld alqqreqqll eslgngtdfs vsssasmdtv 121 tsssssslsv lpsslsvfqn ptdvarsnpk spqkpivrvf lpnkqrtvvp arcgvtvrds 181 lkkalmmrgl ipeccavyri qdgekkpigw dtdiswltge elhvevlenv pltthnfvrk 241 tfftlafcdf crkllfqgfr cqtcgykfhq rcstevplmc vnydqldllf vskffehhpi 301 pqeeaslaet altsgsspsa pasdsigpqi ltspspsksi pipqpfrpad edhrnqfgqr 361 drsssapnvh intiepvnid dlirdqgfrg dggsttglsa tppaslpgsl tnvkalqksp 421 gpqrerksss ssedrnrmkt lgrrdssddw eipdgqitvg qrigsgsfgt vykgkwhgdv 481 avkmlnvtap tpqqlqafkn evgvlrktrh vnillfmgys tkpqlaivtq wcegsslyhh 541 lhiietkfem iklidiarqt aqgmdylhak siihrdlksn niflhedltv kigdfglatv 601 ksrwsgshqf eqlsgsilwm apevirmqdk npysfqsdvy afgivlyelm tgqlpysnin 661 nrdqiifmvg rgylspdlsk vrsncpkamk rlmaeclkkk rderplfpqi lasiellars 721 lpkihrsase pslnragfqt edfslyacas pktpiqaggy gafpvhHuman dihydroorotate dehydrogenase (DHODH) protein (SEQ IN NO:2)   1 klpwrhlqkr aqdaviilgg ggllfasylm atgderfyae hlmptlqgll dpesahrlav  61 rftslgllpr arfqdsdmle vrvlghkfrn pvgiaagfdk hgeavdglyk mgfgfveigs 121 vtpkpqegnp rprvfrlped qavinrygfn shglsvvehr lrarqqkqak ltedglplgv 181 nlgknktsvd aaedyaegvr vlgpladylv vnvsspntag lrslqgkael rrlltkvlqe 241 rdglrrvhrp avlvkiapdl tsqdkedias vvkelgidgl ivtnttvsrp aglqgalrse 301 tgglsgkplr dlstqtirem yaltqgrvpi igvggvssgq dalekiraga slvqlytalt 361 fwgppvvgkv kreleallke qgfggvtdai gadhrr Human BRAF(V600E)(SEQ ID NO: 3)   1 maalsggggg gaepgqalth gdmepeagag agaaassaad paipeevwni kqmikltqeh  61 iealldkfgg ehnppsiyle ayeeytskld alqqreqqll eslgngtdfs vsssasmdtv 121 tsssssslsv lpsslsvfqn ptdvarsnpk spqkpivrvf lpnkqrtvvp arcgvtvrds 181 lkkalmmrgl ipeccavyri qdgekkpigw dtdiswltge elhvevlenv pltthnfvrk 241 tfftlafcdf crkllfqgfr cqtcgykfhq rcstevplmc vnydqldllf vskffehhpi 301 pqeeaslaet altsgsspsa pasdsigpqi ltspspsksi pipqpfrpad edhrnqfgqr 361 drsssapnvh intiepvnid dlirdqgfrg dggsttglsa tppaslpgsl tnvkalqksp 421 gpqrerksss ssedrnrmkt lgrrdssddw eipdgqitvg qrigsgsfgt vykgkwhgdv 481 avkmlnvtap tpqqlqafkn evgvlrktrh vnillfmgys tkpqlaivtq wcegsslyhh 541 lhiietkfem iklidiarqt aqgmdylhak siihrdlksn niflhedltv kigdfglate 601 ksrwsgshqf eqlsgsilwm apevirmqdk npysfqsdvy afgivlyelm tgqlpysnin 661 nrdqiifmvg rgylspdlsk vrsncpkamk rlmaeclkkk rderplfpqi lasiellars 721 lpkihrsase pslnragfqt edfslyacas pktpiqaggy gafpvhHuman dihydroorotate dehydrogenase (DHODH) mRNA - GenBank accessionNM_001361 (SEQ ID NO: 4)   1 gggcttaatg acggaaggag catggcgtgg agacacctga aaaagcgggc ccaggatgct  61 gtgatcatcc tggggggagg aggacttctc ttcgcctcct acctgatggc cacgggagat 121 gagcgtttct atgctgaaca cctgatgccg actctgcagg ggctgctgga cccggagtca 181 gcccacagac tggctgttcg cttcacctcc ctggggctcc ttccacgggc cagatttcaa 241 gactctgaca tgctggaagt gagagttctg ggccataaat tccgaaatcc agtaggaatt 301 gctgcaggat ttgacaagca tggggaagcc gtggacggac tttataagat gggctttggt 361 tttgttgaga taggaagtgt gactccaaaa cctcaggaag gaaaccctag acccagagtc 421 ttccgcctcc ctgaggacca agctgtcatt aacaggtatg gatttaacag tcacgggctt 481 tcagtggtgg aacacaggtt acgggccaga cagcagaagc aggccaagct cacagaagat 541 ggactgcctc tgggggtcaa cttggggaag aacaagacct cagtggacgc cgcggaggac 601 tacgcagaag gggtgcgcgt actgggcccc ctggccgact acctggtggt gaatgtgtcc 661 agccccaaca ctgccgggct gcggagcctt cagggaaagg ccgagctgcg ccgcctgctg 721 accaaggtgc tgcaggagag ggatggcttg cggagagtgc acaggccggc agtcctggtg 781 aagatcgctc ctgacctcac cagccaggat aaggaggaca ttgccagtgt ggtcaaagag 841 ttgggcatcg atgggctgat tgttacgaac accaccgtga gtcgccctgc gggcctccag 901 ggtgccctgc gctctgaaac aggagggctg agtgggaagc ccctccggga tttatcaact 961 caaaccattc gggagatgta tgcactcacc caaggccgag ttcccataat tggggttggt1021 ggtgtgagca gcgggcagga cgcgctggag aagatccggg caggggcctc cctggtgcag1081 ctgtacacgg ccctcacctt ctgggggcca cccgttgtgg gcaaagtcaa gcgggaactg1141 gaggcccttc tgaaagagca gggctttggc ggagtcacag atgccattgg agcagatcat1201 cggaggtgag gacagcgtct gacgggaagc ctgatctgga accttcccaa ggactcaggc1261 aagcctttgt ggctggatca tgagaggagg gactccatct tgagccatgt cccccagcca1321 tggcatggct gcactgtaaa cgccaatcgg ggggtcacca ggatcaaccg caggctttct1381 tcagtccctt ggtcagacca taaactgcat ttttgattct ttgtggattc aaaccctagg1441 atccatcagt cttgcaagga cattgaatat taggaggaaa aagtcatgga aaaaataaag1501 ccatttagaa cctgggtttc aacgctagcc ctttctggtt tgccataggc cctgccaaga1561 tactgcaggt ccatccaggc ctctgctatc tgcatctgca gtgggcttcc caggaacttg1621 actgtctttc atttgatctt tatttttgtt tatttaatat tttaaacttt attttaaaaa1681 tatttcaaac ataagggcgg ggtgtggtgg ctcatgcctg taatcccagc actttgggag1741 gccgaggcgg gcggatcacc tgaggccagg agttggagac cagccaggcc accatggtga1801 aaccctgtct ctaccaaaaa tacaaaaaat tagccagata ttgtggcagg cacttttaat1861 cccagctact cgggaggctg aggcaggaga attgcttgaa cctgggaggc ggaggttgca1921 gtgagccaag attgcaccag tgcactccag cttgggcgac agatcaagac tctgtcacac1981 acaaaacaaa caaacaaatt ttcccgtttc tctctgtccc tttctctata acatggataa2041 aatattcacg tcttatgtat ttattattgc tgagtcattg gtgccttatt cttcagtgtt2101 tcagtctgta tctcctaagc atcctgatgt tagttttttg actataaaat tgacctgcat2161 tctttactag caattcaaac ggtacagcac tcttgaagtg taaacattcc tgttcctcct2221 caccccactc tgcagacatg cctttctgtc tgtcctccca gacttttccc ctgcataaag2281 atgttcattt tgtacataca ctcagacata catgtggctg tattttttgt ataccgattt2341 ctggatggaa tgcacactgt tatctgttta gttactttat gttttcatac aaatgacatc2401 atcccacaaa tagaattctg ccatttaaaa aaaaaaaaHuman BRAF mRNA: v-raf murine sarcoma viral oncogene homolog B1 (BRAF),(SEQ ID NO: 5)   1 cgcctccctt ccccctcccc gcccgacagc ggccgctcgg gccccggctc tcggttataa  61 gatggcggcg ctgagcggtg gcggtggtgg cggcgcggag ccgggccagg ctctgttcaa 121 cggggacatg gagcccgagg ccggcgccgg cgccggcgcc gcggcctctt cggctgcgga 181 ccctgccatt ccggaggagg tgtggaatat caaacaaatg attaagttga cacaggaaca 241 tatagaggcc ctattggaca aatttggtgg ggagcataat ccaccatcaa tatatctgga 301 ggcctatgaa gaatacacca gcaagctaga tgcactccaa caaagagaac aacagttatt 361 ggaatctctg gggaacggaa ctgatttttc tgtttctagc tctgcatcaa tggataccgt 421 tacatcttct tcctcttcta gcctttcagt gctaccttca tctctttcag tttttcaaaa 481 tcccacagat gtggcacgga gcaaccccaa gtcaccacaa aaacctatcg ttagagtctt 541 cctgcccaac aaacagagga cagtggtacc tgcaaggtgt ggagttacag tccgagacag 601 tctaaagaaa gcactgatga tgagaggtct aatcccagag tgctgtgctg tttacagaat 661 tcaggatgga gagaagaaac caattggttg ggacactgat atttcctggc ttactggaga 721 agaattgcat gtggaagtgt tggagaatgt tccacttaca acacacaact ttgtacgaaa 781 aacgtttttc accttagcat tttgtgactt ttgtcgaaag ctgcttttcc agggtttccg 841 ctgtcaaaca tgtggttata aatttcacca gcgttgtagt acagaagttc cactgatgtg 901 tgttaattat gaccaacttg atttgctgtt tgtctccaag ttctttgaac accacccaat 961 accacaggaa gaggcgtcct tagcagagac tgccctaaca tctggatcat ccccttccgc1021 acccgcctcg gactctattg ggccccaaat tctcaccagt ccgtctcctt caaaatccat1081 tccaattcca cagcccttcc gaccagcaga tgaagatcat cgaaatcaat ttgggcaacg1141 agaccgatcc tcatcagctc ccaatgtgca tataaacaca atagaacctg tcaatattga1201 tgacttgatt agagaccaag gatttcgtgg tgatggagga tcaaccacag gtttgtctgc1261 taccccccct gcctcattac ctggctcact aactaacgtg aaagccttac agaaatctcc1321 aggacctcag cgagaaagga agtcatcttc atcctcagaa gacaggaatc gaatgaaaac1381 acttggtaga cgggactcga gtgatgattg ggagattcct gatgggcaga ttacagtggg1441 acaaagaatt ggatctggat catttggaac agtctacaag ggaaagtggc atggtgatgt1501 ggcagtgaaa atgttgaatg tgacagcacc tacacctcag cagttacaag ccttcaaaaa1561 tgaagtagga gtactcagga aaacacgaca tgtgaatatc ctactcttca tgggctattc1621 cacaaagcca caactggcta ttgttaccca gtggtgtgag ggctccagct tgtatcacca1681 tctccatatc attgagacca aatttgagat gatcaaactt atagatattg cacgacagac1741 tgcacagggc atggattact tacacgccaa gtcaatcatc cacagagacc tcaagagtaa1801 taatatattt cttcatgaag acctcacagt aaaaataggt gattttggtc tagctacagt1861 gaaatctcga tggagtgggt cccatcagtt tgaacagttg tctggatcca ttttgtggat1921 ggcaccagaa gtcatcagaa tgcaagataa aaatccatac agctttcagt cagatgtata1981 tgcatttgga attgttctgt atgaattgat gactggacag ttaccttatt caaacatcaa2041 caacagggac cagataattt ttatggtggg acgaggatac ctgtctccag atctcagtaa2101 ggtacggagt aactgtccaa aagccatgaa gagattaatg gcagagtgcc tcaaaaagaa2161 aagagatgag agaccactct ttccccaaat tctcgcctct attgagctgc tggcccgctc2221 attgccaaaa attcaccgca gtgcatcaga accctccttg aatcgggctg gtttccaaac2281 agaggatttt agtctatatg cttgtgcttc tccaaaaaca cccatccagg cagggggata2341 tggtgcgttt cctgtccact gaaacaaatg agtgagagag ttcaggagag tagcaacaaa2401 aggaaaataa atgaacatat gtttgcttat atgttaaatt gaataaaata ctctcttttt2461 ttttaaggtg aaccaaagaa cacttgtgtg gttaaagact agatataatt tttccccaaa2521 ctaaaattta tacttaacat tggattttta acatccaagg gttaaaatac atagacattg2581 ctaaaaattg gcagagcctc ttctagaggc tttactttct gttccgggtt tgtatcattc2641 acttggttat tttaagtagt aaacttcagt ttctcatgca acttttgttg ccagctatca2701 catgtccact agggactcca gaagaagacc ctacctatgc ctgtgtttgc aggtgagaag2761 ttggcagtcg gttagcctgg gttagataag gcaaactgaa cagatctaat ttaggaagtc2821 agtagaattt aataattcta ttattattct taataatttt tctataacta tttcttttta2881 taacaatttg gaaaatgtgg atgtctttta tttccttgaa gcaataaact aagtttcttt2941 ttataaaaa.

The invention claimed is:
 1. A method for treating melanoma in a subjectcomprising: (i) selecting a subject with rising cancer marker proteinlevels or having a family history of cancer or who has had a melanomaremoved surgically; and (ii) administering to a subject in need thereofa therapeutically effective amount of an inhibitor of dihydroorotatedehydrogenase (DHODH) and administering an effective amount of aninhibitor of oncogenic BRAF, wherein the inhibitor of DHODH is selectedfrom the group consisting of:


2. The method of claim 1, wherein the inhibitor of oncogenic BRAF isselected from the group consisting of: a small molecule, a nucleic acidRNA, a nucleic acid DNA, a protein, a peptide, and an antibody.
 3. Themethod of claim 1, wherein the oncogenic BRAF is BRAF(V600E).
 4. Themethod of claim 1, wherein the inhibitor of oncogenic BRAF is selectedfrom the group consisting of: Sorafenib, RAF265, XL281, AZ628,GSK2118436, GDC-0879, PLX4032, and PLX4720.
 5. The method of claim 1,wherein the inhibitor of oncogenic BRAF and inhibitor of dihydroorotatedehydrogenase (DHODH) are administered to the subject sequentially orsimultaneously.
 6. A method for treating melanoma in a subjectcomprising: (i) selecting a subject with rising cancer marker proteinlevels or having a family history of cancer or who has had a melanomaremoved surgically; and (ii) administering to a subject in need thereofa therapeutically effective amount of an inhibitor of dihydroorotatedehydrogenase (DHODH) and administering an effective amount of aninhibitor of oncogenic BRAF, wherein the inhibitor of DHODH is selectedfrom the group consisting of:


7. The method of claim 6, wherein the inhibitor of dihydroorotatedehydrogenase is


8. The method of claim 6, wherein the inhibitor of dihydroorotatedehydrogenase (DHODH) is


9. The method of claim 6, wherein the inhibitor of oncogenic BRAF isselected from the group consisting of: a small molecule, a nucleic acidRNA, a nucleic acid DNA, a protein, a peptide, and an antibody.
 10. Themethod of claim 6, wherein the oncogenic BRAF is BRAF(V600E).
 11. Themethod of claim 6, wherein the inhibitor of oncogenic BRAF is selectedfrom the group consisting of: Sorafenib, RAF265, XL281, AZ628,GSK2118436, GDC-0879, PLX4032, and PLX4720.
 12. The method of claim 6,wherein the inhibitor of oncogenic BRAF and inhibitor of dihydroorotatedehydrogenase (DHODH) are administered to the subject sequentially orsimultaneously.